Antimicrobial resistance

ANTIMICROBIAL
SUSCEPTIBILITY TESTING
 LEARNING OBJECTIVES

• Identify antimicrobial susceptibility testing needs

• Understand standard antimicrobial susceptibility testing

• Interpret antimicrobial susceptibility testing

 LEADING GLOBAL INFECTIOUS DISEASES

S. pneumonia: Up to 55% S. dysentriae: 90% resistance to

resistance to penicillin in cotrimoxazole
some regions S.Typhi: Outbreaks of multi-resistant
strains in 11 countries

M. tuberculosis:
HIV: Report of Multi-drug resistant
resistance to all tuberculosis
marketed agents

P. falciparum:
Chloroquine resistance in
81/92 countries
 ANTIMICROBIAL SUSCEPTIBILITY TESTS
Diffusion
• Kirby-Bauer method
• Stokes method
Dilution:
Minimum Inhibitory Concentration
• Broth dilution
• Agar dilution
Diffusion & Dilution
• E test

Automated Methods

Molecular Methods
 DISK DIFFUSION: KIRBY-BAUER METHOD
• The most widely used antibiotic susceptibility test

• This method relies on the inhibition of bacterial growth

measured under standard conditions

• Mueller-Hinton agar, is uniformly and aseptically inoculated with

the test organism

• Filter paper discs, which are impregnated with a specific

concentration of a particular antibiotic, are then placed on the
medium
 DISK DIFFUSION: KIRBY-BAUER METHOD

Incubate plate
Inoculate the 18-24 hr, 35 C
test organism on Place disks Measure and record
Mueller Hinton on agar plate zone of inhibition
agar plate around each disk

Susceptibility is inferred using standard tables

 DISK DIFFUSION: KIRBY-BAUER METHOD
• The organism will grow on the agar plate while the antibiotic “works” to
inhibit the growth

• If the organism is susceptible to a specific antibiotic, there will be no growth

around the disc containing the antibiotic

• The “zones of inhibition” can be observed and measured to determine the

susceptibility of an organism to a set of antibiotics

• The measurement is compared to the criteria set by the Clinical and

Laboratory Standards Institute (CLSI)

• Based on the criteria, the organism can be classified as being Resistant (R),
Intermediate (I) or Susceptible (S).
 E. Coli (ATCC 25922)
Augmentin (Am) 18-24 mm
Ampicillin (AM)  16-22mm
Cefazolin (CZ) 21-27 mm
Ciprofloxacin (CIP)  30-40 mm
Nitrofurantoin (F/M) 20-25 mm
Sulfisoxazole (GO) 15-23 mm
Trimeth/Sulfa (SX) 23-29 mm
 
 
S. Aureus (ATCC 25923)
      Cefazolin (CZ) 29-35  mm
   Clindamycin (CC2) 24-30 mm
Cefoxitin (FOX)  23-29 mm (Oxacillin)
Erythomycin (E) 22-30 mm
Levofloxacin (LV) 25-30 mm
Nitrofunantoin (F/M) 18-22 mm
Penicillin (P) 26-37 mm
Tetracycline (Te) 24-30 mm
Trimeth/Sulfa (SXT) 24-32 mm
 MUELLER HINTON AGAR (MHA)

• It supports the growth of most of the organisms

• It contains starch. Starch is known to absorb toxins released from bacteria, so

that they cannot interfere with the antibiotics

• It is a loose agar. This allows for better diffusion of the antibiotics

• MHA is low in sulfonamide, trimethoprim, and tetracycline inhibitors

• This reduces the inactivation of sulfonamides and trimethoprim when the

media is used for testing the susceptibility to these antibiotics

STOKES’ COMPARATIVE METHOD

TEST
ORGANISM

CONTROL
ORGANISM

TEST
ORGANISM
 STOKES’ COMPARATIVE METHOD

• In this disc technique, both the test and control organisms are

inoculated on the same plate

• The zone sizes of the test organism are compared directly with

that of the control

• Disadvantage: Only few antibiotics can be tested at a time

 ANTIMICROBIAL GRADIENT TESTING E-TEST

•Epsilometer test (E- test) is an “exponential gradient” method

•Combination of dilution and diffusion of antibiotics into the

medium

•Determines Minimum Inhibitory Concentration (MIC), the lowest

concentration of an antimicrobial that inhibits the visible growth
of a microorganism

•The E-test uses rectangular plastic strips with the predefined,

continuous and exponential gradient of antibiotic concentration
E-TEST
 E-test®

Read plates
after
recommended
Incubation Read MIC
where elipse
intersects
scale
 CRITICAL POINTS IN QUALITY ASSURANCE

1. Culture media: Muller-Hinton, thickness and quality of the

culture media

2. Quality and conservation of the disks

3. Inoculums: Lack of standardization of the inoculums

4. Quality control with standardized strains

5. Incubation condition: Condition and duration of incubation

6. Reading inhibition diameters (accurate measurement)

 STANDARD STRAINS FOR QUALITY ASSURANCE

Precision and accuracy ensured through control strains

• Known susceptibility to antimicrobial agents

Standard strains include

• Staplylococcus aureus ATCC 25923

• Escherichia coli ATCC 25922

• Pseudomonas aeruginosa ATCC 27853

 COMMON INTERPRETATION PROBLEMS

An agar gel that is too thick leads to smaller zones

 COMMON INTERPRETATION PROBLEMS

Problem with the size of the

inoculums

Solution:
Use McFarland 0.5 photometer
Scale -> same tubes
 COMMON INTERPRETATION PROBLEMS

Contamination with another

organism
 COMMON INTERPRETATION PROBLEMS

Bad manipulation

Inoculation of the Muller Hinton

◦ Swabbing

◦ Not by flooding
 COMMON INTERPRETATION PROBLEMS
Problems with E-test reading