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1

ANTIMICROBIAL
SUSCEPTIBILITY
TESTING
PRESENTER : MOHD SHAHZEB
M.Sc. Medical Microbiology
Sharda University, SMS&R
2

Antimicrobial Agents:
 Antimicrobials are the agents that kill (cidal) or
inhibit the growth of microorganisms (static).
 Used to reduce the risk of infection and sepsis.
 Classified in various ways:
3

Classification:
1. Acc. to microorganisms against which they
are used - Antibacterial, antifungal,
Antiparasitic, Antiviral agents, etc.
4

Classification:
2. Acc. to their ability to kill or inhibit the
microorganisms.
5

Classification:
3. Acc. to the source:
 Antibiotics: Natural substances, produced by
certain groups of microorganisms. e.g. penicillium
notatum naturally produces the antibiotic penicillin.
 Chemotherapeutic agents: These agents are
chemically synthesized, e.g. cotrimoxazole, quinolones.
6

Classification:
4. According to their site of action and usage :
i. Disinfectants
ii. Antiseptics
iii. Antibiotics
7

Classification:
5. According to the chemical structure and
mechanism of action.
 Cell wall synthesis inhibition
 Protein Synthesis Inhibition
 Nucleic Acid Synthesis Inhibitors
 Mycolic Acid Synthesis Inhibitors
 Folic acid Synthesis Inhibitors
 Antimicrobial agents that act on cell membrane
8

Cell Wall Synthesis Inhibition
9

Cell Wall Synthesis Inhibition

Cell Wall Synthesis Inhibition
10
11

Protein Synthesis Inhibition
12

Protein Synthesis Inhibition
13

Nucleic Acid Synthesis
Inhibitors
14

Mycolic Acid Synthesis
Inhibitors
15

Folic acid Synthesis Inhibitors
16

Antimicrobial agents that act on
cell membrane
17

Antimicrobial agents
Mechanism:
18

Antimicrobial Resistance
 It refers to development of resistance against
antimicrobial agent by a microorganism.
 They are of two types:
1) Acquired Resistance
2) Intrinsic Resistance
19

Antimicrobial Resistance
Acquired
Resistance
• Bacteria can acquire
resistance through a new
genetic mutation that
helps the bacterium
survive.
OR
• By getting DNA from a
bacterium that already is
resistant
Intrinsic
Resistance
• Innate ability of a bacterium to
resist a class of antimicrobial
agents due to its inherent
structural or functional
characteristics
• e.g. gram-negative bacteria
are resistant to vancomycin
• Klebsiella species are resistant
to ampicillin.

20
Introduction
ANTIMICROBIAL SUSCEPTIBILITY
TESTING
Antibiotic susceptibility testing (AST) is a laboratory test that
determines if a bacteria or fungi is resistant or sensitive
against one or more antibiotics. The test helps identify which
antibiotic will be most effective for treating an infection, and
can also help find a treatment for antibiotic-resistant
infection.

21
 Susceptibility test, main purposes:
 As a guide for treatment
 Sensitivity of a given microorganism to know concentration of drugs.
 Its concentration in body fluids or tissues
 It will control the use of Antibiotics in clinical practice and assist the
clinicians in the choice of drugs for the treatment of infections
 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)

22
Medium
Mueller-Hinton Agar (MHA)
 Mueller-Hinton agar (MHA) is considered as the best medium
to use for routine susceptibility testing of non- fastidious
bacteria for the following reasons:
1. Shows acceptable batch-to-batch reproducibility for
susceptibility testing.
2. Supports satisfactory growth of most non-fastidious
pathogens
3. Minimal inhibitory effect on sulphonamide and
trimethoprim. Hence, these antibiotics are better tested in
MHA than any other media.

23
Mueller-Hinton Agar (MHA)

24
INOCULUM
 Isolated pure colonies of the test organism are inoculated in a suitable liquid
medium (peptone water broth) and incubated at 35-37°C for 4-6 hours.
 Lawn culture: The broth is inoculated on the medium(MHA) by spreading
with sterile swabs.
 Control strains: Similar to the test isolate, the inoculum of control strain should
also be made and tested for AST. The following ATCC (American Type Culture
Collection) strains are used as standard control strains:
 Escherichia coli ATCC 25922
 Pseudomonas aeruginosa ATCC 27853
 Staphylococcus aureus ATCC 25923
 Enterococcus feacalis ATCC 29212.
25

Inoculum Preparation
(NCCLS Reference Method)
 Standardize inoculum
suspension

26
Antibiotic Disk
 Antibiotic disks are available commercially. Sterile filter paper
disks of 6 mm diameter are impregnated with standard
quantity of antibiotic solution.
 Choice of Antibiotic Disk:
 Antibiotics should likely to be used for therapy
 Organism against which the drug has to be tested
 Local prescribing habits of the antimicrobial agents
 Resistant pattern of the locally prevalent pathogens
 Cost, toxicity and spectrum of activity of an antimicrobial
agent for the management of illness in a particular patient.

27
 Drugs for routine susceptibility tests:
 Tier 1: Drugs that are available in most hospitals and for
which routine testing should be carried out for every
strain
 Tier 2: Drugs that are reported 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
28

Routine Susceptibility Tests
 Disk diffusion
methods
 Dilution Test
 Etest

29
Disk Diffusion Methods
 Disk diffusion tests are the most widely used
method.
 Suitable for rapidly growing pathogenic bacteria.
 Uses filter paper disks impregnated with appropriate
concentration of the antibiotic solution.
 There are two methods:
 Kirby-Bauer Disk Diffusion Method
 Stokes Disk Diffusion Method

30
Kirby-Bauer Disk Diffusion Method
 Firstly we do lawn culture by the inoculum that we already
made.
 After drying the surface of agar plate antibiotics will be applied
using sterile forceps or multi-disk dispenser.
 Disks should not be placed closer than 20 mm on the MHA
plate.
 Ordinarily, max up to 6 disks can be applied on a 100 mm
plate.
 The plates are then incubated at 37°C for 16-18 hours.
 The zones of complete growth of inhibition around each of the
disks are measured using a ruler or Vernier caliper.
31

Remove sample Lawn Culture
rim the plate
with the
swab

32

33
Kirby-Bauer Disk Diffusion Method
Antibiotic Disk
34

Incubate Overnight
35

Read the Results with Precision

36
Stokes Disk Diffusion Method
 The MHA plate is divided into two halves. The test strain and
control strain are inoculated separately in each half.
 An uninoculated gap of 2-3 mm wide should separate the
test and the control area on which the antibiotic disks are
applied.
 The plates are then incubated at 37°C for 16-18 hours.
 The sensitivity report is prepared by comparing the zones of
inhibition of control and test strain
37


38
Direct Disk Diffusion Test
 Direct disk diffusion test may be performed when results are
required urgently and single pathogenic bacterium is
suspected in the specimen.
 The specimen is directly inoculated uniformly on the surface
of an agar plate and the antibiotic disks are applied. The results
of the primary test should be verified by testing the isolates
subsequently.
 Test has no use when mixed growth of different bacteria is
suspected in the specimen, e.g. pus, stool, sputum, etc.
39
Disc Diffusion Method
Reporting the Results
 Interpretation of results
 By comparing with the diameters acc.to
“CLSI (Clinical and laboratory Standards
Institute) Guidelines”
 Susceptible
 Intermediate susceptible
 Resistant
40
Disc Diffusion Method
 Measurement of the diameters of inhibition
zone
 Measure from the edge where the growth stop,
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.

41
Factors Affecting Size of Zone
of Inhibition
 Inoculum density
 Timing of disc application
 Temperature of incubation
 Incubation time
 Larger zones with light
inoculum and vice versa
 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 o
C
 Ideal 16-18 hours; less time
does not give reliable results

42
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 zone.
 Avoids overlapping of
zones
43
Factors Affecting Size of Zone of
Inhibition
 Potency of antibiotic
discs
 Composition of
medium
 Acidic pH of medium
 Alkaline pH of
medium
 Reading of zones
 Deterioration in contents leads
to reduced size
 Affects rate of growth,
diffusion of antibiotics and
activity of antibiotics
 Tetracycline, novobiocin,
methicillin zones are larger
 Aminoglycosides,
erythromycin zones are larger
 Subjective errors in
determining the clear edge
44
Broth Dilution Method
Dilution Tests
Agar Dilution Method
Macro Broth Dilution
Micro Broth Dilution
45

 The antimicrobial agent is serially diluted, each
dilution is tested with the test organism for
antimicrobial susceptibility test and the MIC is
calculated.
 MIC (minimum inhibitory concentration) is the
lowest concentration of an antimicrobial agent that
will inhibit the visible growth of a microorganism
after overnight incubation.
Dilution Tests

46
Dilution Method
 Minimum Bactericidal Concentration
(MBC): The lowest concentration of antimicrobial
agent that allows less than 0.1% of the original
inoculum to survive.
47

 Macro Broth Dilution: Making dilutions of
antibiotic in broth Mueller-Hinton.
 Each tube is inoculated with a fixed amount of
suspension of the test organism.
 Controls: no inoculum, no antibiotic.
 No visible growth MIC
 Sub culturing of non-turbid tubes, overnight
 No Growth (bacterial count)  MBC
Broth Dilution Method
48

49
Micro broth Dilution Method
 Micro dilution plates:
 96 wells/plate: simultaneously performed with
many tests organisms/specimens,.
 Manually prepared
 Commercially prepared
 Frozen or Dried
 Consistent performance but high cost
 May suffer from degradation of antibiotic during
shipping and storage.

64 32 16 8 4 2
ug/ml
Micro broth Dilution
51
Agar Dilution Method
 The serial dilutions of the drug are prepared in molten
agar and poured into petri dishes.
 One concentration of antibiotic/plate
 Possible for several different strains/plate
 It directly measures the MBC; there is no need of sub­
culturing.
64 ug/ml 32 ug/ml 16 ug/ml

52
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

53
A Steers-Foltz replicator
54

Agar Dilution Method
55

Epsilometer or E-test
 A well established AST method in microbiology laboratories
around the world.
 Quantitative method of detecting MIC by using the principles of
both dilution and diffusion methods.
 Uses an absorbent strip containing predefined gradient (serial
dilution) of antibiotic concentration immobilized along its length.
 It is applied to a lawn inoculum of a bacterium, an elliptical zone
of inhibition is produced surrounding the strip.
56

57

Clinical Conditions where MICs are more
Useful
 Endocarditis
 Meningitis
 Septicemia
 Osteomyelitis
 Immunosuppressed patients (HIV, cancer,
etc.)
 Prosthetic devices
58

VITEK 2 Automates Reporting
of Resistance
 Vitek 2 is a fluorescence-based
test that uses the broth
microdilution minimum
inhibitory concentration (MIC)
technique to identify bacteria and
determine their susceptibility to
antibiotics.
 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.
59

Essentials of Medical Microbiology, Apurba S Sastry, 3rd edition
References
60

Thank You

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Antimicrobial Susceptibility Testing: Methods, Challenges, and Innovations

  • 1. 1  ANTIMICROBIAL SUSCEPTIBILITY TESTING PRESENTER : MOHD SHAHZEB M.Sc. Medical Microbiology Sharda University, SMS&R
  • 2. 2  Antimicrobial Agents:  Antimicrobials are the agents that kill (cidal) or inhibit the growth of microorganisms (static).  Used to reduce the risk of infection and sepsis.  Classified in various ways:
  • 3. 3  Classification: 1. Acc. to microorganisms against which they are used - Antibacterial, antifungal, Antiparasitic, Antiviral agents, etc.
  • 4. 4  Classification: 2. Acc. to their ability to kill or inhibit the microorganisms.
  • 5. 5  Classification: 3. Acc. to the source:  Antibiotics: Natural substances, produced by certain groups of microorganisms. e.g. penicillium notatum naturally produces the antibiotic penicillin.  Chemotherapeutic agents: These agents are chemically synthesized, e.g. cotrimoxazole, quinolones.
  • 6. 6  Classification: 4. According to their site of action and usage : i. Disinfectants ii. Antiseptics iii. Antibiotics
  • 7. 7  Classification: 5. According to the chemical structure and mechanism of action.  Cell wall synthesis inhibition  Protein Synthesis Inhibition  Nucleic Acid Synthesis Inhibitors  Mycolic Acid Synthesis Inhibitors  Folic acid Synthesis Inhibitors  Antimicrobial agents that act on cell membrane
  • 10.  Cell Wall Synthesis Inhibition 10
  • 16. 16  Antimicrobial agents that act on cell membrane
  • 18. 18  Antimicrobial Resistance  It refers to development of resistance against antimicrobial agent by a microorganism.  They are of two types: 1) Acquired Resistance 2) Intrinsic Resistance
  • 19. 19  Antimicrobial Resistance Acquired Resistance • Bacteria can acquire resistance through a new genetic mutation that helps the bacterium survive. OR • By getting DNA from a bacterium that already is resistant Intrinsic Resistance • Innate ability of a bacterium to resist a class of antimicrobial agents due to its inherent structural or functional characteristics • e.g. gram-negative bacteria are resistant to vancomycin • Klebsiella species are resistant to ampicillin.
  • 20.  20 Introduction ANTIMICROBIAL SUSCEPTIBILITY TESTING Antibiotic susceptibility testing (AST) is a laboratory test that determines if a bacteria or fungi is resistant or sensitive against one or more antibiotics. The test helps identify which antibiotic will be most effective for treating an infection, and can also help find a treatment for antibiotic-resistant infection.
  • 21.  21  Susceptibility test, main purposes:  As a guide for treatment  Sensitivity of a given microorganism to know concentration of drugs.  Its concentration in body fluids or tissues  It will control the use of Antibiotics in clinical practice and assist the clinicians in the choice of drugs for the treatment of infections  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)
  • 22.  22 Medium Mueller-Hinton Agar (MHA)  Mueller-Hinton agar (MHA) is considered as the best medium to use for routine susceptibility testing of non- fastidious bacteria for the following reasons: 1. Shows acceptable batch-to-batch reproducibility for susceptibility testing. 2. Supports satisfactory growth of most non-fastidious pathogens 3. Minimal inhibitory effect on sulphonamide and trimethoprim. Hence, these antibiotics are better tested in MHA than any other media.
  • 24.  24 INOCULUM  Isolated pure colonies of the test organism are inoculated in a suitable liquid medium (peptone water broth) and incubated at 35-37°C for 4-6 hours.  Lawn culture: The broth is inoculated on the medium(MHA) by spreading with sterile swabs.  Control strains: Similar to the test isolate, the inoculum of control strain should also be made and tested for AST. The following ATCC (American Type Culture Collection) strains are used as standard control strains:  Escherichia coli ATCC 25922  Pseudomonas aeruginosa ATCC 27853  Staphylococcus aureus ATCC 25923  Enterococcus feacalis ATCC 29212.
  • 25. 25  Inoculum Preparation (NCCLS Reference Method)  Standardize inoculum suspension
  • 26.  26 Antibiotic Disk  Antibiotic disks are available commercially. Sterile filter paper disks of 6 mm diameter are impregnated with standard quantity of antibiotic solution.  Choice of Antibiotic Disk:  Antibiotics should likely to be used for therapy  Organism against which the drug has to be tested  Local prescribing habits of the antimicrobial agents  Resistant pattern of the locally prevalent pathogens  Cost, toxicity and spectrum of activity of an antimicrobial agent for the management of illness in a particular patient.
  • 27.  27  Drugs for routine susceptibility tests:  Tier 1: Drugs that are available in most hospitals and for which routine testing should be carried out for every strain  Tier 2: Drugs that are reported 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
  • 28. 28  Routine Susceptibility Tests  Disk diffusion methods  Dilution Test  Etest
  • 29.  29 Disk Diffusion Methods  Disk diffusion tests are the most widely used method.  Suitable for rapidly growing pathogenic bacteria.  Uses filter paper disks impregnated with appropriate concentration of the antibiotic solution.  There are two methods:  Kirby-Bauer Disk Diffusion Method  Stokes Disk Diffusion Method
  • 30.  30 Kirby-Bauer Disk Diffusion Method  Firstly we do lawn culture by the inoculum that we already made.  After drying the surface of agar plate antibiotics will be applied using sterile forceps or multi-disk dispenser.  Disks should not be placed closer than 20 mm on the MHA plate.  Ordinarily, max up to 6 disks can be applied on a 100 mm plate.  The plates are then incubated at 37°C for 16-18 hours.  The zones of complete growth of inhibition around each of the disks are measured using a ruler or Vernier caliper.
  • 31. 31  Remove sample Lawn Culture rim the plate with the swab
  • 33.  33 Kirby-Bauer Disk Diffusion Method Antibiotic Disk
  • 35. 35  Read the Results with Precision
  • 36.  36 Stokes Disk Diffusion Method  The MHA plate is divided into two halves. The test strain and control strain are inoculated separately in each half.  An uninoculated gap of 2-3 mm wide should separate the test and the control area on which the antibiotic disks are applied.  The plates are then incubated at 37°C for 16-18 hours.  The sensitivity report is prepared by comparing the zones of inhibition of control and test strain
  • 38.  38 Direct Disk Diffusion Test  Direct disk diffusion test may be performed when results are required urgently and single pathogenic bacterium is suspected in the specimen.  The specimen is directly inoculated uniformly on the surface of an agar plate and the antibiotic disks are applied. The results of the primary test should be verified by testing the isolates subsequently.  Test has no use when mixed growth of different bacteria is suspected in the specimen, e.g. pus, stool, sputum, etc.
  • 39. 39 Disc Diffusion Method Reporting the Results  Interpretation of results  By comparing with the diameters acc.to “CLSI (Clinical and laboratory Standards Institute) Guidelines”  Susceptible  Intermediate susceptible  Resistant
  • 40. 40 Disc Diffusion Method  Measurement of the diameters of inhibition zone  Measure from the edge where the growth stop, 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.
  • 41.  41 Factors Affecting Size of Zone of Inhibition  Inoculum density  Timing of disc application  Temperature of incubation  Incubation time  Larger zones with light inoculum and vice versa  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 o C  Ideal 16-18 hours; less time does not give reliable results
  • 42.  42 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 zone.  Avoids overlapping of zones
  • 43. 43 Factors Affecting Size of Zone of Inhibition  Potency of antibiotic discs  Composition of medium  Acidic pH of medium  Alkaline pH of medium  Reading of zones  Deterioration in contents leads to reduced size  Affects rate of growth, diffusion of antibiotics and activity of antibiotics  Tetracycline, novobiocin, methicillin zones are larger  Aminoglycosides, erythromycin zones are larger  Subjective errors in determining the clear edge
  • 44. 44 Broth Dilution Method Dilution Tests Agar Dilution Method Macro Broth Dilution Micro Broth Dilution
  • 45. 45   The antimicrobial agent is serially diluted, each dilution is tested with the test organism for antimicrobial susceptibility test and the MIC is calculated.  MIC (minimum inhibitory concentration) is the lowest concentration of an antimicrobial agent that will inhibit the visible growth of a microorganism after overnight incubation. Dilution Tests
  • 46.  46 Dilution Method  Minimum Bactericidal Concentration (MBC): The lowest concentration of antimicrobial agent that allows less than 0.1% of the original inoculum to survive.
  • 47. 47   Macro Broth Dilution: Making dilutions of antibiotic in broth Mueller-Hinton.  Each tube is inoculated with a fixed amount of suspension of the test organism.  Controls: no inoculum, no antibiotic.  No visible growth MIC  Sub culturing of non-turbid tubes, overnight  No Growth (bacterial count)  MBC Broth Dilution Method
  • 49. 49 Micro broth Dilution Method  Micro dilution plates:  96 wells/plate: simultaneously performed with many tests organisms/specimens,.  Manually prepared  Commercially prepared  Frozen or Dried  Consistent performance but high cost  May suffer from degradation of antibiotic during shipping and storage.
  • 50.  64 32 16 8 4 2 ug/ml Micro broth Dilution
  • 51. 51 Agar Dilution Method  The serial dilutions of the drug are prepared in molten agar and poured into petri dishes.  One concentration of antibiotic/plate  Possible for several different strains/plate  It directly measures the MBC; there is no need of sub­ culturing. 64 ug/ml 32 ug/ml 16 ug/ml
  • 52.  52 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
  • 55. 55  Epsilometer or E-test  A well established AST method in microbiology laboratories around the world.  Quantitative method of detecting MIC by using the principles of both dilution and diffusion methods.  Uses an absorbent strip containing predefined gradient (serial dilution) of antibiotic concentration immobilized along its length.  It is applied to a lawn inoculum of a bacterium, an elliptical zone of inhibition is produced surrounding the strip.
  • 57. 57  Clinical Conditions where MICs are more Useful  Endocarditis  Meningitis  Septicemia  Osteomyelitis  Immunosuppressed patients (HIV, cancer, etc.)  Prosthetic devices
  • 58. 58  VITEK 2 Automates Reporting of Resistance  Vitek 2 is a fluorescence-based test that uses the broth microdilution minimum inhibitory concentration (MIC) technique to identify bacteria and determine their susceptibility to antibiotics.  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.
  • 59. 59  Essentials of Medical Microbiology, Apurba S Sastry, 3rd edition References