ACT antimicrobial susceptibility testing, inoculation and drug sensitivity

Prepared by Dr. Sadaf Konain Ansari for 2nd year BDS

Antimicrobial Antimicrobial Susceptibility Testing Susceptibility Testing

(AST)(AST)

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 bacteria’s susceptibility to particular antimicrobial agents is uncertain

Susceptibilities NOT performed on bacteria that are predictably susceptible to antimicrobials

Ex. Group A Strep

Dr.Sadaf Konain Ansari

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


Factors to Consider When Determining Whether Testing is Warranted (cont’d)

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


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


Selecting Antimicrobial Agents for Testing and Reporting (cont’d)

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


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


Example of Drug FormularyDrug 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


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


Definitions (important to remember)

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.


Definitions (cont’d)

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)


Standardization of Antimicrobial Susceptibility Testing

Inoculum Preparation

Use 4-5 colonies NOT just 1 colony

Inoculum Standardization

using 0.5 McFarland standard


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


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


Disk Diffusion/ Kirby-Bauer (cont’d)

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


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


Automated Antimicrobial Susceptibility Test

Methods

Detect growth in micro volumes of broth with various dilutions of antimicrobials

Detection via photometric, turbi-dimetric, or fluoro-metric methods

Types

BD Phoenix

Microscan Walkaway

TREK Sensititre

Vitek 1 and 2


Automated Antimicrobial Susceptibility Test Methods

AdvantagesIncreased reproducibility

Decreased labor costs

Rapid results

Software

Detects multi-drug resistances

ESBLs

Correlates bacterial ID with sensitivity

DisadvantagesCost


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


The Superbugs (important to remember)

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


The Superbugs: The 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


Controlling the Superbugs

Lab’s Role

Recognize and report isolates recovered from clinical specimens

Methods for identification include automated systems and screening agars


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?doc=CNL_CLN_PRD_G_PRD_CLN_22

http://www.cdc.gov/std/gonorrhea/lab/diskdiff.htm

http://www.who.int/drugresistance/Antimicrobial_Detection/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. Dr.Sadaf Konain Ansari


Exercise Questions

ESKAPEPathogens of Highest Concern The most serious, life- threatening infections are caused by a group of drug- resistant bacteria that the Infectious Diseases Society of America (IDSA) has labeled the "ESKAPE" pathogens, because they effectively escape the effects of antibacterial drugs

What are ESKAPE


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.

What stand for MIS, and MBC?


Common Questions:

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.

What stands for S, I and R?


Common Questions:

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ACT antimicrobial susceptibility testing, inoculation and drug sensitivity