This document provides information on antimicrobial susceptibility testing using disk diffusion methods. It discusses the importance of AST for treating infectious diseases and monitoring antimicrobial resistance. The Kirby-Bauer disk diffusion method is described in detail, including media preparation, inoculum standardization, disk and antibiotic solution preparation, quality control strains, incubation, reading zones of inhibition, and interpreting results according to CLSI guidelines. Special considerations are given to organisms like MRSA, VISA, and inducible clindamycin resistance in Staphylococci.

1. ANTIMICROBIAL
SUSCEPTIBILITY TESTING –
DISK DIFFUSION METHODS

2. INTRODUCTION
Antimicrobial Susceptibility Test is very important
for treating infectious diseases and monitoring
antimicrobial resistance in various pathogens.
It is essential that the reports are
relevant,
timely
interpreted correctly
to ensure Quality Control.

3.  To guide the clinician- selection of antibiotics
 To accumulate epidemiological information
on the resistance of microorganisms of
public health
 importance within the community.

4. DEFINITION
AST :
It is a determination of least amount of an
antimicrobial chemotherapeutic agent that will
inhibit the growth of microorganism invitro.
Quality control :
A process in the laboratory designed to monitor the
analytical phase of testing procedure to ensure
that tests are working properly.

5. AST methods
a. Disk diffusion method:
1. Kirby Bauer method
2. Stokes method
b. MIC:
1. Broth dilution method
2. Agar dilution method
c. E-test

6. Diffusion-Kirby Bauer method
 Principle
Paper disks impregnated with antimicrobial agent are
placed on agar medium uniformly seeded with the test
organism.
A concentration gradient of the antibiotic is formed by
diffusion from the disk and the growth of the test
organism is inhibited at a distance form the disk (that
is related among other factor) to the susceptibility of
the organism

7. Medium
 According to CLSI (clinical laboratory standard institute)
Muller Hinton Agar - Non fastidious organism
 Temperature - 45°C to 50°C
 Thickness 4mm
 PH 7.2 – 7.4
 Moisture
 Storage: 5 days at 2-8°C
 Prolonged storage causes – dehydration of the media
 MHA Plates wrapped in air tight plastic bags and
refrigerated – 2 weeks

8. Media used
Muller Hinton Agar
 It is best for non fastidious organism
 It shows acceptable batch to batch
reproducibility
 It has low thymidine content.
(Increased thymidine antagonise the activity of
sulphonamides)
 Reverse the inhibitory effect of SXT – lesser or
no zone-falls resistant report
 To check QC – ATCC 29212
E.faecalis – SXT ->20mm

9. MHBA ( 5% sheep blood agar )
Strept. Pneumoniae
Beta strept, alpha strept, non haemolytic strept
MHCA & HTM
Haemophilus spp
GC agar
Gonococci
Media used contd

10. Antibiotics
Commercial disk
Whenever we receive the antibiotics check the label,
Mfg date, Exp date and Lot no.
It should be checked with ATCC strains
Stored at -20°C or -70°C and at 4°C – 8°C
Routine use keep at 4°C – 8°C
Paper disk (In-house)
Whatmann filter paper No. 2 is used
Diameter 6mm with regular edges
Sterilize by hot air oven at 160°C for 1hour
Do not use irregular edged and charred disk

11. Antibiotic solution preparation
 It is always prepared from pure substance
 Stock made concentrations depending on disk strength
 Some antibiotics dissolved in organic solvent and others
in sterile distilled water
 Use only minimum volume of organic solvent to stabilize
the antimicrobial powder
 After preparing the solution should checked with ATCC
strains
 Prepared antibiotics are aliquote into 6-7 ml in tubes
 Lesser amount – improper delivery of antibiotic

12. Antibiotic solution preparation
 Eg; Ampicillin – Needed concentration-2000µg/ml(DD
strength 10µg/ml)
 1mg=1000µg/ml
 2mg=2000µg/ml
 20mg=2000µg/10ml
 200mg=2000µg/100ml
 Volume stock (in ml) =weight(mg) x potency of
antibiotic(µg) /needed concentration(µg)
 Obtaining satisfactory results, dispense 10ml into 20ml
sterile tube
 Store it at -20°C for six months

13. Inoculum
 Turbidity standard for inoculum preparation
 McFarland Standard – BaSO4
0.5 - 2 x 108 - for GNB and fast growing organism
1.0 - 3 x 108 - for Gram positive cocci

14. PREPARATION OF CULTURE
Select 10 morphologically identical well isolated
colonies
Inoculate in 1.5ml NB
Incubate for 2hrs

15. Adjust opacity – McFarland's standard
0.5 for Gram Negative bacilli
1 for Gram Positive cocci

16. INOCULATION
 Marking the plates
 Six antibiotics – 85 to 90mm petridishes
 Two antibiotics – QC
 Streaking the plates
( within 15 mins after opacity adjusted )
 Placing the disks
In-between two disk – 24mm
Periphery to the disk – 15mm
Overlapping of zone of inhibition should be
avoided
Time duration – 15mins
Loop 2mm diameter it delivers 0.005ml
Ensure complete contact to the agar surface
disk should not be relocated

17. INCUBATION & ATMOSPHERE
MHA plates are incubated at 37°C for 16-18hrs
MHBA, HTM are incubated at 37°C with 5% CO2
incubator

18. ATCC control strains for AST
QUALITY CONTROL
To check the quality of the medium
Potency of the antibiotic
Technical error
When ever we receive new drug or media

19. Quality control (QC)
 QC - A procedure which ensures that the
performance of a test/procedure is reliable
 QC in AST - Testing a standard strain of known susceptibility
to the antimicrobial agent tested
 Goal of QC - Accuracy and reproducibility

20. ATCC control strains for AST
 Eg: ATCC 25923 Staph aureus (beta lactamase negative,
oxacillin - susceptible)
 ATCC 27853 Pseudomonas aeruginosa (for
aminoglycosides)
 ATCC 25922 E.coli (beta lactamase negative)
 ATCC 35218 E.coli (beta lactamase positive)
 ATCC 29212 E.faecalis (for checking of Thymidine level of
MHA)
 ATCC 700603 Kleb. Pneumoniae (ESBL-positive)
 ATCC 49619 Strept. Pneumoniae (oxa – R)
 ATCC 49247 H.influenzae
 ATCC 49766 H.influenzae
Procedure for performing QC

21. READING
Each zone size is interpreted according to the
organism by reference in the CLSI guidelines
RESISTANCE :resistant , to indicate that the
bacteria can not be inhibited by the antibiotics.
INTERMEDIATE : intermediate , to indicate that
the bacteria can be inhibited by the high dose of
antibiotics.
SUSCEPTIBLE :susceptible, to indicate that the
bacteria can be inhibited by the normal dose of
antibiotics

23. READING AND INTERPRETATION
Only pure growth is considered for reading
Inoculum should be adequate
There should not be any misplacing of antibiotics
 Quality control strains should be in expected
ranges (guided by CLSI)

24.  Acidic p H of medium
 Alkaline p H of medium
 Addition of thymidine to
medium
 Low content of thymidine
Less action
aminoglycoside,
quinolones and macrolides,
excess activity of tetra
More activity of
Aminoglycosides,
quinolones and macrolides
Lesser activity of tetra
Decrease activity of SXT –
resistant zone
ATCC 29212 E.faecalis – SXT
more than – 20mm
satisfactory

25. Magnesium + Calcium (cation)
Excess : reduce zone size for aminoglycoside
Low : increase zone size for aminoglycoside
Zinc
Excess : reduce zone size for carbapenems
Lesser : increase zone size in carbapenems

26. Larger zone of inhibition
Light inoculum
Error in inoculum preparation
Depth of the medium is thin
MHA is nutritionally unacceptable
Smaller zone of inhibition
heavy inoculum
Error in inoculum preparation
Depth of the medium is thick

27. One or more zone too small or too large zone
Measurement error
Transcription error
Random defective disk
Disk not pressed firmly to the agar surface
One QC strain is out of range but other QC strain are within
range for the same antibiotic
One may be the better indicator of QC problem
Two QC strain is out of for the same antibiotic
Problem with the disk

28. Cont..
 Reading……
 Zone of inhibition measured in diameter or radius with transparent
ruler

29.  Cont…
 Colonies within the zones
of inhibition
 Zones overlap
 Zones indistinct
 Mixed culture
 Resistant mutants within
the zone.
 disks too close together
 Poorly streaked plate

30. • Cont….
Double zone
Proteus swarming – ignore
Fastidious organism – eg, beta Strept,
S.pneumoniae – zone of inhibition
not by haemolysis
Co-trimoxazole reading

31. Precautions
Ampicillin is always R to Klebsiella and Aeromonas
spp
Nitrofurantoin S to E.coli R to Proteus and
Klebsiella
Cefoxitin R – MRSA
Cefpodoxime R – ESBL in GNB
Imipenem and meropenem R – CRO

32.  Cont….
Vancomycin and teicoplanin resistant – VRE
Alert forms – HICC, MS office, respective
units/wards
S.typhi and S. paratyphi A newer guideline for
ciprofloxacin – >31 is S and MIC by E.test
Oxacillin R S.pneumoniae do penicillin MIC

33. ADVANTAGES
Technically simple to perform
Reproducible reagents are inexpensive
Does not require any special equipments
Easily understood by clinicians
Flexible regarding the selection of antibiotics

34. STOKES METHOD
Procedure

35. Stokes method
Susceptible – zone size of the test strain is larger
than or equal to control strain
Resistant – zone size of the test strain is smaller
than 2mm
Intermediate – zone size of the test strain is 2-
3mm smaller than that of the control strain
Advantages
Both control and test organism is same environment
Disadvantages
 2 to 4 antibiotics in one plate is tested
Laborious

36. Things need to
be known

37. Organisms requiring special considerations
• Emergence of resistance:
• Staphylococci:
• Methicillin resistant S.aureus:
• Oxacillin and other penicilinase resistant penicillin such as
methicillin, cloxacillin constitute the drug of choice for
Staphylococcal infections.
• Methicillin is no longer the agent of choice for testing
and treatment.
• The penicillin binding protein which has low affinity for
binding all beta-lactam drugs is encoded by the gene
mec A

38. Contd....
• mec A is responsible for resistance to methicillin
and other beta lactam antibiotics
• mec A encodes penicillin binding proteins 2a,
which differs from other penicillin binding protein
as its active site does not bind methicillin or other
beta lactam antibiotics
• Penicillin binding protein 2a can continue to
catalyze the transpeptidation reaction required
for peptidoglycan, enabling the cell wall synthesis
in the presence of antibiotics

39. Contd....
• Consequence of the inability of PBP2a to interact
with beta lactams
• Acquition of mecA confers resistance to all beta
lactam antibiotics in addition to methicillin
• MRSA is significant in hospital acquired and
community associated infections
• Drug of choice – vancomycin and teicoplanin –
injectable
• Rifampacin and linezolid – oral drug
• Topical application – bacitracin, chlorohexidine,
mupirocin

40. Detection methods for MRSA
• Cefoxitin DD – surrogate marker
• Because cefoxitin serves to induce greater
expression of PBP2a in mec A containing strains of
Staphylococci and also function as test reagent to
detect resistant.
• Oxacillin screen plate
– MHA with 4%Nacl + 6mg per ml
– Spot inoculate – incubate at 350C
– More than one colony indicates oxacillin resistant
 Molecular detection by PCR can be performed

41. • CLSI recommends cefoxitin for specific break
points interpretative criteria for S.aureus and
Coagulase neg Staph.
• Cefoxitin – zone of inhibition can be easily read
than oxacillin DD.
Break points for DD interpretation
Cefoxitin Oxacillin
Resistance Susceptible Resistance Intermediate Susceptible
S.aureus 21 22 10 11-12 13
CONS 24 25 17 - 18

42. Cefoxitin vs Oxacillin
• Cefoxitin
• Stable drug
• Requires 16-18hrs
incubation at 370C
• No supplement is
necessary
• Clear zone of inhibition
and wider range of
interpretative criteria
• Oxacillin
• Degradation on storage
• Requires 24hrs incubation
at 350 C
• 2-5% Nacl is added
• Narrow range of
interpretative criteria
hence zone of inhibition is
measured using
transmitted light

43. Vancomycin resistance or diminished
susceptibility in S.aureus
• Strains with reduce susceptibility to vancomycin have been
called vanco intermediate S.aureus (VISA) or glycopeptide
intermediate (GISA)
• Between 2002-2005 five different strain of MRSA were
detected for the first time with vancomycin resistant.
• The first MRSA isolate with more subtle diminished
susceptibility to vancomycin with MIC value 8mg per ml
(intermediate)
• Although still uncommon both vanco(R) S.aureus and VISA
are of great concern because vanco is the drug of choice for
MRSA

44. DETECTION METHODS
• Vancomycin DD
• Vancomycin MIC
• Vancomycin agar screen test
– Brain heart infusion agar with vancomycin 6mg per ml

45. Inducible clindamycin resistant in
Staphylococci:
• Two different resistant mechanisms confers
macrolide resistance (e.g. erythromycin)
• The erm gene codes for methylation of 23S r RNA
which results in resistant to erythromycin and
either inducible or constitutive resistant to
clindamycin.
• The msrA gene codes for an efflux mechanisms
which results in resistance to erythromycin but
susceptible to clindamycin.

46. D-Zone positive Negative

47. • D zone test for inducible clinda resistance to be
performed before reporting clindamycin
• For the D zone test erythromycin and clindamycin
disk to be placed 15-26 mm edge to edge on MHA
by usual DD test.
• Incubation-16-18hrs at 37c.
• Flatening of the clinda zone between the 2 disk –
indicates the isolate has inducible clindamycin
resistant because of erm gene
• No flatening the isolate is erythromycin resistant
(due to msrA).

48. • D zone positive- clindamycin resistant
• D zone negative- clindamycin susceptible
• Both erythromycin and clindamycin resistant- clindamycin
resistant.
Vancomycin resistant Enterococci:
• E. faecium and the E.faecalis are most common resistant to
vancomycin and teicoplanin
• Six different types of vancomycin resistance
• Van A and B are most commonly encountered
• Van A – resistance to both vancomycin and teicoplanin
• Van B - resistance to vancomycin and susceptible to
teicoplanin

49. VRE mechanism
• Alteration to the terminal amino acid residues of
the NAM/NAG-peptide subunits
• The D-alanyl-D-lactate variation results in the loss
of one hydrogen-bonding interaction
• This loss of just one point of interaction results in
a decrease in affinity between vancomycin and
peptide

50. VRE detection
• Disk diffusion
• MIC by broth dilution, agar dilution and E-test
• Vancomycin agar screen plate
• BHIA with 6mg vancomycin can be used for
Enterococci and Staphylococci
• Drugs – daptomycin, linezolid, quinipristin
dalfopristin

51. High level aminoglycoside resistance
• Enterococci are inherently resistant to the
concentration of aminoglycoside producing their use
as single agent for treatment of enterococcal
infections
• This low level resistance is due to the poor drug
uptake by the enterococcal cells.
• However enterococci develop high level
aminoglycoside resistance in which the particular
aminoglycoside does not demonstrate synergism
with the cell wall active agent penicillin or ampicillin.

52. • High level aminoglycoside resistant in
enterococci is usually the result of enzyamtic
inactivation of the drugs.
• Detection by
DD – Gentamicin 120µg
MIC - Gentamicin 500µg

53. ESBL
• The major mechanism of resistant to β-lactam antimicrobial
agent in Gram negative bacilli is production of β-lactamase
enzyme because of their increased spectrum activity.
• ESBL are a group of plasmid mediated diverse complex and
rapidly evolving enzymes that are posing a major therapeutic
challenge in the treatment of hospitalized and community
based patients.
• Infections caused by ESBL strains from UTI to life threatening
sepsis.

54. • Β-lactamase these enzymes share the ability to hydrolyse
These cephalosporins include cefotaxime,ceftriaxone,
and ceftazidime, as well as monobactam aztreonam.
• ESBL – producing organsims exhibit co-resistance to many
other class of antibiotics.
• Because of inoculum effect and substrate specificity –their
detection is also major challenge.
• But now CLSI gives the guideline for detection of ESBL in
Klebsiella pneumoniae, K.oxytoca, E.coli and Pr.mirabilis.
• ESBL are β-lactamase capable of confering bacterial
resistance to the penicillin, I, II and III generation of
cephalosporins and aztreonam

55. β lactamases
Restricted
spectrum
β lactamases
ESBL
AmpC β
lactamases
CTX-M OXA
Serine MBL
Class B
Class A OXA
Class D
OthersClassical
TEM-1 & 2,
SHV-1
TEM-3,
SHV-2
Over
65
types
11, 14,
15, 16,
17
CMY, LAT,
FOX
KPC,
SME,
IMI
23, 24,
40, 51,
58
Carbapenemases
IMP,
VIM,
NDM

56. ESBL detection tests - Phenotypic
Screening Confirmatory
1) Disk diffusion method
2) Dilution method – MIC
1) Diffusion methods
- Double disc
- Combination disc
- E-test
2) Dilution method – MIC

57. Icil
Confirmatory
tests
Double disc approximation test
Qualitative only
CLSI combination disc test
Qualitative and quantitative
CTX CTX + CL
CZD CZD + CL
cefotaxime ceftazidime
Amox + clav

58. CLSI ESBL confirmatory tests
interpretation
For E. coli, Klebsiella, and P. mirabilis
MIC test ≥3 two-fold concentration decrease in MIC of
cefotaxime/ceftazidime +/- clavulanate 4 μg/ml
Disk test ≥5-mm increase in zone diameter for
cefotaxime/ceftazidime +/- clavulanate 10 μg

59. Icil
AmpC Beta lactamases
• Chromosomal mediated in Enterobacter, Serratia,
Citrobacter, Morganella, Providencia
• Plasmid-mediated in E. coli and Klebsiella
Emergence predominantly in community-acquired
infections
• Co-resistance to aminoglycosides, SXT, quinolones
TEM, SHV CTX-M OXA AmpC
Cefpodoxime R R R R
Clavulanate S S R R
Cephamycins S S S R
Cefepime R R R S

60. Drug of Choice
• Carbapenems (Impenem, meropenem)
• Colistin, polymyxin, tigecycline for serious
infections
• Co-trimoxazole, nitrofurantoin, fosfomycin,
gentamycin, amikacin and inhibitor combinations
for uncomplicated infections.

61. Carbapenemase
• Carbapenems – Highest class of beta lactam agent
current available, eg, imipenem, meropenem,
ertapenem
• Carbapenem resistance to all beta lactam antibiotics
such as penicillin, cephalosporins, monobactams and
carbapenems
• Carbapenem resistance due to
Production of carbapenemases
Excess production of ESBL, porin loss, increased
efflux pumps
• Carbapenemases are beta lactamase enzyme coded
by plasmids

62. • In GNB most commonly encountered are
• Klebsiella pneumoniae carabapenemase (KPC) -
Serine in their active site
• Metallo beta lactamases – Zinc in their active site
• Metallo beta lcatamases – R to all beta lactams
but S to monobactam
• KPC – R to all beta lactam / beta lactamase
inhibitor

63. Detection methods
• DD – imipenem, meropenem
• MIC – broth, agar dilutions
• E-test
Modified Hodge test
• Lawn culture of E. coli ATCC 25922 - 1/10 of 0.5
McFarland
• ERT10 μg
• Inoculate cultures as shown in figure
• edge of disk to periphery

64. CONCLUSION
• AST is very important for the clinician to treat the
patient with appropriate antibiotics.
• Formulation of antibiotic policy
• Surveillance of resistance
– In community
– Hospital out breaks
• Lab to upgrade its own good standard
• Ensures accuracy, reliability reproducibility of the
test performed.