The document provides an overview of antimicrobial susceptibility testing (AST) methods, including diffusion methods (like Kirby-Bauer), dilution methods (agar and broth), and automated systems, as well as methods for testing antiviral and antifungal susceptibility. It details the importance of AST in determining appropriate antibiotic therapy and assessing the effectiveness of treatments against bacterial, viral, and fungal infections. Additionally, it highlights the standardization efforts and references established for best practices in susceptibility testing.

1. SUSCEPTIBILITY TESTING
BY:ANJALI SHARMA (M.Sc.)
DEPARTMENT OF MICROBIOLOGY

2. CONTENTS:
• INTRODUCTION
• ANTIBACTERIAL SUSCEPTIBILITY TESTING
1. Diffusion method
• Strokes disc diffusion
• Kirby bauer disc diffusion
2. Dilution method
• Agar dilution
• Broth dilution
3. Diffusion and dilution
• E- test

3. • ANTIVIRAL SUSCEPTIBILITY TESTING
1. Phenotypic susceptibility assay
2. Genotypic susceptibility assay
3. Plaque reduction assay
4. Dye uptake assay
5. DNA hybridization assay
6. Pyrosequencing
• ANTIFUNGAL SUSCEPTIBILITY TESTING

4. INTRODUCTION
• AST ( ANTIMICROBIAL SUSCEPTIBILITY TESTING) is used to determine how
effective antibiotic therapy is against a bacterial infection.
• AST will control the use of antibiotics in clinical practice.
• For e.g. : BACITRACIN DISC TEST – For identification of S.pyrogenes (as this spp.
Is sensitive to minute concentration (0.02 micro gm) of BACITRACIN -gives positive
test.

5. IMPORTANCE/GOALS OF AST :
• The identification of relevant antibiotics to specific pathogens in exudates and
body fluids collected from patients.
• Sensitivity tests down to determine the degree of sensitivity or resistance of
pathogens isolated from patient to an appropriate range of antimicrobial drugs .
• Assay of concentration of an administered drug in blood or body fluid of patient
required to control the schedule of dosage.
• The RESULTS of AST should be combined with clinical information and experience
when selecting the most appropriate antibiotic for patient .
• The raw data are either in form of a [INHIBITORY ZONE ] OR [MIC] . Typically , raw
data are interpreted and reported out as;

6. • SUSCEPTIBLE /SENSITIVE ORGANISMS :
• This category implies that the organisms is inhibited by the serum concentration of
antimicrobial agent i.e. achieved by using the usual dosage recommended for the
type of infection present .
• INTERMEDIATE ORGANISMS :
• Implies that the organisms are inhibited only by the maximum recommended
dosage .
• Strains that show “intermediate susceptibility” to a more toxic antibiotic .
• E.G.: aminoglycoside that can not be used at a higher dosage .
• RESISTANT ORGANISMS :
• Implies that the organisms are resistant to usually achieveable serum drug
concentration of antimicrobial agent.

7. • THESE TESTS ARE PERFORED IN-VIVO AND UNDER STANDARDIZED
CONDITIONS:
1. STANDARD MEDIA : Muller-Hinton agar or broth.
2. STANDARD INOCULUM : Isolation and standardizing the bacteria suspend by
Macfarland standards [ Mcf =1.5microMF ].
• Macfarland standard : standard unit to measure turnbidity of bacterial suspension .
3. INCUBATION TIME AND TEMPERATURE : (35-37) ,16-18 hrs .
4. VOLUME OF MEDIA USED : 0.5 Mcf std.

8. TYPES OF ANTI-BACTERIAL
SUSCEPTIBILITY TESTING :
DILUTIO
N
DIFFUSION
AST
[DIFFUSION
AND
DILUTION]
AGAR DIUTION
BROTH DILUTION
STROKES DISC
DIFFUSION
KIRBY BAUER DISC
DIFFUSION
E-TEST

9. DILUTION METHODS :
1. BROTH DILUTION METHODS :
• First performed in test tubes , now in – shallow wells of std . Plates .
• MEDIA: muller – hinton broth , nutrient broth .
• ANTIBIOTIC CONCENTRATION : 0.1-0.2 micro gm / m litre
• INCUBATION TIME : 16-20 hrs.
• E.G. : UTI – E.coli –overnight broth culture in peptone - std. inoculum.
• In MICROBROTH DILUTION METHOD , We will take some amount o fnutrient broth
in series of test tubes .
i. Serial dilution of concentration of antibiotic prepared in nutrient broth .
ii. And a NEGATIVE CONTROL with no antibiotic .
iii. Add 1 ml of std. inoculum to all test tubes .
iv. Incubate overnight at 37 Celsius.

11. • CONTROL : max growth (max. turbidity).
• As the concentration of antibiotics increases , the turbidity decreases .
• At a spwcific concentration – no turbidity -( MIC ).
• If the whole process sis done in petridish , it is called as MICROBROTH DILUTION
METHOD .
• ADVANTAGE : Quantitative method [MIC can be done ].
• DISADVANTAGE : 1) fastidious organisms can not be done .
2) Cumbersome procedure.

12. • AGAR DILUTION METHOD :
• MEDIUM : cation adjusted muller-hinton agar
• ANTIBIOTIC CONCENTRATION : 0.1-0.2 micro gm / ml
• INCUBATION TIME : 16-20 hrs ,(35-37 )celcius.
• PROCEDURE :
i. Pour the media in sterile petri plates .
ii. Prepare serial dilution of antibiotic concentration .
iii. Spread it through sterile glass spreader .
iv. Look for lowest concentration of antibiotic prevent the appearance of colonies
[MIC].
• ADVANTAGES: 1) fastidious bacteria can be tested
2) Can be used for anaerobes
3)MIC can be determined .
• DISADVANTAGES : 1) Cumbersome procedure .

14. DISK DIFFUSION METHOD :
• KIRBY-BAUER DIFFUSION METHOD :
• Simple and quick agar diffusion test.
• Developed by – William M. Kirby and A.W. Bauer .
• Most common method in use .
• MEDIUM : Muller-hinton agar (5% blood added if required ).
• STANDARD INOCULUM : 0.5 McF turbidity.
• PROCEDURE :
i. Using a sterile cotton swab , the inoculum is spread onto MH agar medium (lawn culture of
bacteria )
ii. Discs (impregnated with single standardized concentration of antibiotics )are placed on the
swab inoculated petri dish .
iii. Overnight incubation (16-20)hrs at 25 celcius .
iv. ZONE OF INHIBITON SEEN AROUND EACH DISC .

15. v. Measure and compare with standardized tables .
• ADVANTAGES : very easy to do .
• DISADVANTAGES : MIC cannot be determined , qualitative method .

16. • STOKES DIFFUSION METHOD :
• Only followed in certain European countries .
• On the same plate – antibiotic disc , control strain and test strain placed .
• Incubated at same condition .
• Therefore , no need of any tables to compare .
• ADVANTAGES : easy to do .
• DISADVANTGAES : MIC can’t be determined .

17. DILUTION –DIFFUSION METHOD:
• EPSILOMETER TEST /E-TEST :
• Combination of both diffusion and dilution test.
• MEDIUM : muller –hinton agar.
• STANDARD INOCULUM : 0.5 McF turbidity .
i. Instead of discs , PLASTIC STRIPS impregnated with graded concentration of
antibiotics (serial dilution) along its length .
ii. INCUBATION : 24-48 hrs
iii. ELLIPTICAL ZONE of INHIBITION can be seen .
iv. The concentration at which zone of inhibition intersect the plastic strip will
determine the MIC .

18. • ADVANTAGES : Quantitative test , mic determined .
• DISADVANTAGES : sometimes results may confuse – go fr dilution or diffusion
method instead .

19. SERUM KILLING POWER :
• SERUM =blood plasma – clotting proteins .
• Used to determine effectiveness of chemotherapeutic agent .
• From patient’s blood sample (serum ) – when on antibiotic therapy.
i. suspension of bacterial pathogens .
ii. Known quantity of patient’s serum (from freshly collected tubes ) .
iii. Incubated at 35 Celsius .
iv. NO GROWTH : antibiotics are working (effective ).
v. TURBIDITY : not working ( ineffective ).

20. AUTOMATED METHODS :
• Modern , efficient and less expensive method for AST .
i. Sample (suspension of measured quantity of microbe )
ii. Wells on plastic tray containing chemical reagent .
iii. Tests carried out in incubation chamber .
iv. Incubated overnight (1-20) hrs , 37 celsius .
v. Growth and results are taken by computer .
vi. RESULTS : 1) AFTER OVERNIGHT INCUBATION.
2) FOR SLOW GROWING ORGANISMS – 48 HRS .
• ADVANTAGES : 1) plastic trays are available for wide variety of microorganisms – gm (+) ,
gm (-) , anaerobic , yeasts .
2)Many organisms from different patients can be inoculated at the same time .

21. TYPES OF ANTIVIRAL
SUSCEPTIBILITY TESTING :
ANTIVIRAL SUSCEPTIBILITY TESTING
PHENOTYPIC SUSCEPTIBILITY
ASSAY
GENOTYPIC SUSCEPTIBILITY
ASSAY

22. AVST INTRODUCTION :
• Very few standards have been established for AVST BY CLINICAL and LABORATORY STANDARDS
INSTITUTE .
• The final results of AVST is determined b various variables .
• These variables includes :
1. Cell line used to grow virus .
2. Viral inoculum litre .
3. Incubation time of culture .
4. Concentration range of antiviral drug tested .
5. Reference strains
6. Assay methods .
7. End point criteria .
8. Calculation of end point .
9. Interpretation of end point .

23. METHODS OF ANTIVIRAL
SUSCEPTIBILITY TESTING :
• PURPOSE :
1. To evaluate new antiviral chemoprophylaxis .
2. To test for cross-resistance .
3. To determine how frequently drug – resistance viral mutation occur.

24. PHENOTYPIC SUSCEPTIBITLITY
ASSAY :
• Uses variety of end points measurements .
• Used to determine , whether a virus is inhibited by an antivirak drug or
demonstrates drug resistance .
• END POINTS : 1) reduction in number of plaques .
2) Inhibition of viral DNA synthesis .
3) Reduction of viral protein synthesis .
4) Reduction in enzymatic activity.
• Measure inhibitory effect (zone of inhibition ) on entire viral population in clinical
isolate .

25. • ADVANTAGES : 1) used to assess combined effect of multiple resistance mutation on
drug susceptibility .
2) Useful for assaying viruses [such as ; hepaitis B virus , HIV -1 , HCMV ]
• DISADVANTAGES : 1) labor intensive .
2) Expensive .
3) long exposure time / long turn around time .

27. GENOTYPIC SUSCEPTIBILITY ASSAY:
• Uses RTPCR to detect genes , which can mutate , coupled with genetic sequence or
molecular sequence .
• To determine genetic mutation with resistance occurred .
• USES : 1)DNA sequence by automated sequence method .
2)PCR amplification .
3) Restriction enzyme digestion of products .
4)DNA probes .
• RAPID TECHNIQUE : because isolation of culture is not necessary .
• Response to antiviral agent also measured by QUANTITATIVE METHOD of viral load in
patients serum .
• E.G.: hepatitis c virus , CMV .

28. VIRAL GROWTH APPEARS
ANTIVIRAL DRUG IS NOT
WORKING , VIRUS IS NOT
SUSCEPTIBLE
NO VIRAL GROWTH
APPEARS
DRUG IS WORKING ,
VIRUS SUSCEPTIBLE

29. • ADVANTAGES : 1) less exposure time .
2) Less expensive .
• DISADVANTAGES : 1) only detect defined viral mutation .
• After G.S.A.
• The viral load should significantly diminish following the addition of antiviral agent
to which virus is susceptible .
• Using molecular testing , such as quantitative PCR , to measure the amount of
virus present in serum is a surrogate test for resistance to antiviral agent .
• The viral load rises quickly when resistance appears .

31. PLAQUE REDUCTION ASSAY :
• Standard method of antiviral susceptibility assay .
• PRINCIPLE : determine of viral plaque formation in presence of antiviral agent .
• CONCENTRATION OF ANTIVIRAL DRUG :
• Inhibit plaque formation by 50% is [ IC 50].
• [50% inhibitory concentration +50% effective concentration ].

32. DYE UPTAKE ASSAY :
• Used for years .
• Determine viral cell lytic activity .
• Virus- in prescence of antiviral drug .
• Alive and viable cell.
• Takes vital cell [neutral red ] + HSV infection .
• RESULTS : dye bound to in comparison to dye bound to uninfected cell .
• This determines the extent of viral lytic activity .
• Drug concentration that inhibits viral lytic activity by 50% is IC50

33. DNA HYBRIDISATION ASSAY :
• Measure effect of antiviral reagents on synthesis of viral DNA .
• Measures how much viral DNA is produced in absence of antiviral agent .
• IC50 is calculated from these comparisons .
• USED FOR : HSV , VZV , HCMV .

34. PYROSEQUENCING :
• Most important method .
• Sequence based detection method .
• Allow rapid , accurate quantification of sequence variation .
• Allow rapid acquisition of short reads [100-200 bp ] of genomic sequence to
identify known mutation .
• Based on – technique of detection of released [Ppi] pyrpphosphate during DNA
synthesis .
• Visible light produced during reactions .
• Visible light strength generated directly proportional to number of nucleotide
incorporated into final product .

35. TYPES OF PYROSEQUENCING :
PYROSEQUENCING
SOLID PHASE LIQUID PHASE
INVOLVES 3 ENZYME
SYSTEM
1) IMMOBILIZED DNA
2) REMOVAL OF EXCESS
SUBSTRATE
3) NUCLEOTIDE
ADDITION
3 ENZYME SYSTEM + 4
NUCLEOTIDE DEGRADING
ENZYME

36. • The availability of specific antiviral agent has added importance and urgency to the
need for laboratory diagnosis of viral infection .
• Resistance of virus establish an exact etiologic diagnosis is usually needed to justify
use of these expensive and sometimes toxic agents.

37. ANTIFUNGAL SUSCEPTIBILITY
TESTING:
• Antifungal susceptibility testing are designed to provide information that will allow
the physician to select the appropriate Anti- fungal agent useful for treating a
specific infection.
• But A.F.S.T. are not progressed as compared to A.B.S.T.
• An effort is going into standardization for a method that is reproducible between
the laboratories.
• All the techniques variables and end-point are standardized and efforts are
underway to develop interpretative guidelines for different antifungal agents.
• CLSI (CLINICAL LABORATORY STANDARD INSTITUTE) and NCCLS (NATIONAL
COMMITTEE FOR CLINICAL LABORATORY STANDARD) , provides documents and
set standard for A.F.S.T on their website at www.nccls.org.

38. • GUIDELINES FOR A.F S.T. :
1. M27-A2 document - reference method for (broth dilution A.F.S.T of yeast )
appropriate standard.
2. M38-A - reference method for A F.S.T. Of filamentous fungi.
3. M44-A – reference method for antifungal disk diffusion susceptibility testing of
yeast .
• A.F ST. METHODS are still evolving .
• A.F.ST. are costly and time consuming.

39. REFERENCES :
• cle/49/11/1749/344384
• https://en.m.wikipedia.org/wiki/Antibiotic_sensitivity_testing
• https://www.synbiosis.com/application-notes/antibiotic-susceptibility-test-ast/
• https://clinicalgate.com/antiviral-therapy-susceptibility-testing-and-
prevention/#:~:text=The%20purpose%20of%20antiviral%20susceptibility,drug-
resistance%20viral%20mutations%20occur.
• https://www.uptodate.com/contents/antifungal-susceptibility-
testing#:~:text=The%20need%20for%20reproducible%2C%20clinically,problem%2
0%5B1-4%5D.