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Antimicrobial Susceptibility Tests

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Antimicrobial Susceptibility Tests

provide information for selection ofan appropriate agent for antimicrobial therapy

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Antimicrobial susceptibility testsMinimum inhibitory concentration [MIC]

The smallest concentration of antibiotic that inhibits the growth of organism

Liquid media (dilution) allows MIC estimationSolid media (diffusion)

Disk diffusion (Kirby-Bauer)

E-tests

Allows MIC estimation Beta lactamase production: quick screening method

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AST Methods Interpretation

agar disk diffusion method provides qualitative interpretive category results of susceptible, intermediate, and resistant

microdilution and agar gradient diffusion methods provide a quantitative result, a minimum inhibitory concentration

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AST Methods

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Dilution in liquid broth Tubes containing increasing antibiotic concentrations Incubation during 18 hr at 37°C

0 (Control) 0,25 0,50 1 2 4 8 mg/l

MICBacterial growth Inhibition

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Kirby-Bauer disc testingAntibiotic-impregnated discs placed on an agar plate at theinterface between test organism and susceptible control

organismResulting zones of inhibition compared, use of controls Susceptibility is inferred (standard tables)

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E-testPlastic strips with a predefined

gradient of One antibiotic One antifungal

Only one manufacturer One strip per antibioticWide range of antibioticsEasy to useStorage at -20°CShort shelf life, expensive

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Reading E-tests

Susceptible < 1

Resistant > 4 ug/ml

Ciprofloxacin for Yersinia pestis

Intermediate 1-4 ug/ml

Upper reading

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Where errors can occur in susceptibility testing

media antimicrobials inoculum incubation equipment interpretation

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Agar disk diffusion method

Medium Mueller Hinton 4 mm thickness pH 7.2 to 7.4

Antibiotic storage -20oC minimum disks temperature

Inoculum McFarland 0.5 (108 bacteria/mL)

Incubator temperature 35oC

atmosphere ambient air

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Reference Strains

E. coli ATCC 25922 S. aureus ATCC 25923 P. aeruginosa ATCC 27853

QC organisms must be obtained from reputable source

Use specific QC organisms to test different groups of “drug-bug” combinations

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BAP are enriched, differential media used to isolate fastidiousorganisms and detect hemolytic activity. β-hemolytic activity will show lysis and complete digestion of red blood cell contents surrounding colony

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Hemolyses of Streptococcus spp. (left) α-hemolysis (S. mitis); (middle) β-hemolysis (S. pyogenes); (right) γ-hemolysis (= non-hemolytic, S. salivarius)

17Macconkey agar with lactose (left) and non-lactose (right) fermenters

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Selection of a Colony to Test

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MacFarland 0.5 and Adjusted Test Organism

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Disk Susceptibility Testing Problems

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Disk Susceptibility Testing Problems

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Measuring Conditions

RulerCalipers

read with good light, and from the back of the platezone size reading is drug specific magnification may helpmillimeters matter

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Etest – antimicrobial gradient method

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Patient results may be incorrect if:

the organism was misidentified a clerical error was made inappropriate choice of antimicrobials

were tested and reported the wrong patient’s sample was examined the wrong test was ordered the sample was not preserved properly

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Critical points in quality assurance1. Culture media: Muller-Hinton2. Reagents: disks3. Size of the inoculums4. Incubation condition5. Control with reference strains6. Reading inhibition diameters (accurate

measurement)7. Knowledge of staff

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Standard strains for quality assurance

Precision and accuracy ensured through control strainsKnown susceptibility to antimicrobial agents

Standard strains includeStaplylococcus aureus ATCC 25923 Escherichia coli ATCC 25922 Pseudomonas aeruginosa ATCC 27853

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The main concept is the “clinical categorisation" Strains are sorted according to level of Minimal

Inhibitory Concentration (MIC) versus reference breakpoints

c and C are the minor and major breakpoints

Susceptible Intermediate

Resistant

MIC < c ≤ MIC < C ≤ MIC

Interpretation

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Understanding breakpointsWords of laboratory specialists

It is not possible to work aloneBreakpoints are the expression of a consensus

among the scientific community at a given time in a country

Breakpoints are determined using two approaches

Pharmacological concept Epidemiological concept

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0

10

20

30

40

50

60

0.01 0.03 0.06 0.12 0.25 0.5 1 2 4 8 16 32 64 128

MICc

Wild type

Inherited resistance

mechanism

C

The epidemiological concept for breakpoints

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The pharmacological concept for breakpoints

The concentration range tested for a drug and theinterpretative criteria for various categories are based onextensive studies that correlate with

Serum achievable levels for each antimicrobial agent Particular resistance mechanisms Successful therapeutic outcome

In practice situations the entire range may not be used fordecision making and therefore the concept of breakpointconcentration

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From breakpoints to interpretation

Measuring antimicrobial sensitivity of a strain isolated from a patient, to determine its status as S, I or R is an individual problemDefining the status of a bacterial species or genus is an epidemiological problem distributed across time and space that requires monitoring

MIC ≤ c Sensitive strain

MIC > C Intermediate strain

c < MIC ≤ C Resistant strain

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Host factors affecting treatment

Diffusion in tissuesSerum protein bindingDrug interactionsImmune systemMultiple simultaneous infectionsVirulence of organismSite and severity of infection

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Interpreting intermediate resistance

Sometime the agent can still be usedHigher doses required to ensure efficacy Agent may be efficacious if concentrated in

vivo in an infected body fluid (e.g., urine)

Sometimes there is uncertainty Intermediate resistance may represent a

“buffer” zone that prevents strains with borderline susceptibility from being incorrectly categorized as resistant

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Common interpretation problemsResults depends on the technique used Many factors influence results

Lack of standardization of the inoculumsThickness and quality of the culture

mediaQuality and conservation of the disksWuality control with standardized strains Condition and duration of incubation

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An agar gel that is too thick leads to smaller zones

Common interpretation problems

Source: http://www.who.int/csr/resources/publications/drugresist/WHO_CDS_CSR_RMD_2003_6/en/

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Common interpretation problems

Problem with the size of the inoculums

Solution: Use McFarland 0.5 photometer Scale -> same tubes

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Common interpretation problems

Contamination with another organism

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Common interpretation problems

Bad manipulationInoculation of the

Muller Hinton Swabbing Not by flooding

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Problems with E-test reading

Common interpretation problems

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Cost of anti-microbial resistanceCheap antimicrobials become ineffectiveIndividual treatment failureProlonged illness, hospitalizationNeed to switch to more expensive, complex drugs that

areoften not even available in resource-poor settingsNeed to develop new antimicrobialsGood antimicrobial susceptibility testing saves lives andmoney

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WHO Global Principles For The Containment of AntimicrobialResistance In Animals Intended for Food

Department for Communicable Diseases Surveillance and Response

World Health Organization

Report of a WHO Consultation

with the participation of the Food and Agriulture Organization and the

Office International des Epizooties

Geneva, Switzerland

5 – 9 June 2000

WHO/CDS/CSR/APH/2000.4

Distr. : General

English only

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Developed by the Department of Epidemic and Pandemic Alert and Response of the World Health Organization with assistance from:

European Program for Intervention Epidemiology Training

Canadian Field Epidemiology Program

Thailand Ministry of Health

Institut Pasteur

Antimicrobial resistance

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References Manual for the laboratory Identification and

Antimicrobial Susceptibility Testing of Bacterial Pathogens of Public Health Importance in the Developing World WHO/CDS/CSR/RMD/2003.6 http://www.who.int/csr/resources/publications/drugresist/WHO_CDS_CSR_RMD_2003_6/en/