Antimicrobial Susceptibility Testing (AST)

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Antimicrobial Susceptibility Testing (AST)

• MLAB 2434 Microbiology
• Keri Brophy-Martinez

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Reasons and Indications for Antimicrobial
Susceptibility Testing (AST)

• Goal
• Offer guidance to physician in selecting
  effective antibacterial therapy for a pathogen in
  a specific body site
• Performed on bacteria isolated from clinical
  specimens if the bacterias susceptibility to
  particular antimicrobial agents is uncertain
• Susceptibilities NOT performed on bacteria that
  are predictably susceptible to antimicrobials
• Ex. Group A Strep

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Factors to Consider When Determining Whether
Testing is Warranted

• Body site of infection
• Susceptibility not performed on bacteria isolated
  from body site where they are normal flora
• Ex. Susceptibility for E. coli is NOT performed
  when isolated from stool, but IS performed when
  isolated from blood

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Factors to Consider When Determining Whether
Testing is Warranted (contd)

• Presence of other bacteria and quality of
  specimen
• Ex. Two or more organisms grown in a urine
  specimen
• Host status
• Immunocompromised patients
• Allergies to usual antimicrobials

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Selecting Antimicrobial Agents for Testing and
Reporting

• Clinical Laboratory Standards Institute (CLSI)
• Develop standards, methods, QC parameters, and
  interpretive criteria for sensitivity testing
• If necessary, can alter the breakpoints of the
  SIR ( susceptible, intermediate, resistant) based
  on emerging resistance

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Selecting Antimicrobial Agents for Testing and
Reporting (contd)

• There are approximately 50 antibacterial agents
• Follow CLSI recommendations
• Each laboratory should have a battery of
  antibiotics ordinarily used for testing
• Drug formulary decided by medical staff,
  pharmacists, and medical technologists

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Selection of Test Batteries

• Generally, labs choose 10-15 antibiotics to test
  susceptibility for GP organisms and another 10-15
  for GN organisms
• Too many choices can confuse physicians and be
  too expensive
• Primary objective
• Use the least toxic, most cost-effective, and
  most clinically appropriate agents
• Refrain from more costly, broader-spectrum agents

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Example of Drug Formulary
Drug Enterococcus Staphylococcus spp.
Ampicillin X
Cefazolin X
Clindamycin
Erythromycin X
Linezolid X X
Oxacillin X
Penicillin G X X
Rifampin X
Streptomycin-2000 X
Tetracycline X X
Trimeth/ Sulfa X
Vancomycin X X
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Example of Drug Formulary
Drug Enterobacteriaceae Ps. aeruginosa
Ampicillin X
Piperacillin/ Tazo. X X
Cefepime X X
Imipenem X X
Gentamycin X X
Tobramycin X X
Ciprofoxacin X X
Levofloxacin X X
Nitrofurantoin X
Trimethoprim/Sulfa X
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Definitions

• Minimum inhibitory concentration(MIC)
• Lowest concentration of an antimicrobial agent
  that visibly inhibits the growth of the organism.
• Minimum bactericidal concentration (MBC)
• Lowest concentration of the antimicrobial agent
  that results in the death of the organism.

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Definitions (contd)

• Susceptible S
• Interpretive category that indicates an organism
  is inhibited by the recommended dose, at the
  infection site, of an antimicrobial agent
• Intermediate I
• Interpretive category that represents an organism
  that may require a higher dose of antibiotic for
  a longer period of time to be inhibited
• Resistant R
• Interpretive category that indicates an organism
  is not inhibited by the recommended dose, at the
  infection site, of an antimicrobial agent.

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Methods of Performing AST

• Agar dilution method
• Broth macrodilution / Tube dilution
• Broth microdilution
• Disk diffusion method
• Gradient diffusion method (E-Test)

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Standardization of Antimicrobial Susceptibility
Testing

• Inoculum Preparation
• Use 4-5 colonies NOT just 1 colony
• Inoculum Standardization
• using 0.5 McFarland standard

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Methods of Performing AST

• Agar Dilution
• Dilutions of antimicrobial agent added to agar
• Growth on agar indicates MIC
• Broth macrodilution/Tube Dilution Tests
• Two-fold serial dilution series, each with 1-2
  mL of antimicrobial
• Too expensive and time consuming
• Microdilution Tests
• plastic trays with dilutions of antimicrobials

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Disk Diffusion/ Kirby- Bauer

• Procedure
• Use a well-isolated, 18-24 hour old organism
• Transfer organism to a broth
• Either tryptic soy/sterile saline
• Ensure a turbidity of 0.5 McFarland
• Inoculate MH agar by swabbing in three different
  directions Lawn of growth
• Place filter paper disks impregnated with
  anitmicrobial agents on the agar
• Invert and incubate for 16-18 hours at35 oC in
  non-CO2

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Disk Diffusion/ Kirby-Bauer (contd)

• During incubation, drug diffuses into agar
• Depending on the organism and drug, areas of no
  growth form a zone of inhibition
• Zones are measured to determine whether the
  organism is susceptible, intermediate, or
  resistant to the drug

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E- test/ Gradient Diffusion Method

• MIC on a stick
• Plastic strips impregnated with antimicrobial on
  one side
• MIC scale on the other side
• Read MIC where zone of inhibition intersects E
  strip scale

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Automated Antimicrobial Susceptibility Test
Methods

• Detect growth in microvolumes of broth with
  various dilutions of antimicrobials
• Detection via photometric, turbidimetric, or
  fluorometric methods
• Types
• BD Phoenix
• Microscan Walkaway
• TREK Sensititre
• Vitek 1 and 2

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Automated Antimicrobial Susceptibility Test
Methods

• Advantages
• Increased reproducibility
• Decreased labor costs
• Rapid results
• Software
• Detects multi-drug resistances
• ESBLs
• Correlates bacterial ID with sensitivity
• Disadvantages
• Cost

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Quality Control in Susceptibility Testing

• Reflects types of patient isolates range of
  susceptibility
• Frequency of quality control depends on method,
  CLSI, or manufacturer
• Reference strains of QC material
• American Type Culture Collection(ATCC)
• E. coli ATCC 25922
• S. aureus ATCC 25923

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The Superbugs

• Organisms resistant to previously effective drugs
• MRSA
• methicillin-resistant Staphylococcus aureus
• mecA gene codes for a PBP that does not bind
  beta-lactam antibiotics
• Resistant to oxacillin
• Vancomycin
• VRE Enterococcus species
• VISA/VRSA- Staphylococcus aureus

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The SuperbugsThe Beta-Lactamases

• Gram negative rods that have genes on chromosomes
  that code for enzymes against certain
  antimicrobials
• ESBLs-extended spectrum beta lactamase
• Resistant to extended spectrum cephalosporins,
  penicillins, aztreonam
• Examples E. coli, Klebsiella
• Carbapenemases (CRE)
• Klebsiella pneumoniae- KPC- Class A
• Class B (NDM, VIM, IMP)- metallo beta lactamases
• Resistant to penicillins, cephalosporins,
  carbapenems, and aztreonam
• Cephalosporinases
• AmpC enzyme
• inducible
• SPACE organisms

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Controlling the Superbugs

• Labs Role
• Recognize and report isolates recovered from
  clinical specimens
• Methods for identification include automated
  systems and screening agars

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Controlling the Superbugs

• Role of Health Care Workers/Facilities
• Hand hygiene with the use of alcohol-based hand
  rubs or soap and water after patient care
• Contact precautions for patients identified as
  colonized or infected with a superbug
• Healthcare personnel education about the methods
  of transmission, contact precautions, and proper
  use of hand hygiene
• Minimization of invasive devices (catheters,
  etc.)
• Proper administration of antimicrobial agents
  where therapy is selected for susceptible
  organisms for the proper duration

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References

• http//www.biomerieux-diagnostics.com/servlet/srt/
  bio/clinical-diagnostics/dynPage?docCNL_CLN_PRD_G
  _PRD_CLN_22
• http//www.cdc.gov/std/gonorrhea/lab/diskdiff.htm
• http//www.who.int/drugresistance/Antimicrobial_De
  tection/en/index.html
• Kiser, K. M., Payne, W. C., Taff, T. A. (2011).
  Clinical Laboratory Microbiology A Practical
  Approach . Upper Saddle River, NJ Pearson
  Education.
• Mahon, C. R., Lehman, D. C., Manuselis, G.
  (2011). Textbook of Diagnostic Microbiology (4th
  ed.). Maryland Heights, MO Saunders.
• Murray, P. R. (2013, May). Carbapenem-resistant
  Enterobacteriaceae what has happened, and what
  is being done. MLO, 45(5), 26-30.