Module 7 Flashcards by Taylor Horlock

When is it adequate to just identify the organism?

When the organism is universally susceptible

When standardized susceptibility test methods aren’t available

Ex) anaerobic bacteria

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What is the goal of susceptibility/sensitivity testing?

Provide a list of effective antimicrobials for that specific organism.

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What pharmacological knowledge must be applied to the results of a susceptibility test?

Is the antimicrobial effective at the pH of the body site?

Will it penetrate the site of infection?

Is it toxic to the host?

Is it cost effective?

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What are the three conventional methods of performing susceptibility testing?

Broth dilution

Agar dilution

Disc diffusion

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Why is standardized bacteria inoculum used and what is the concentration?

Improves accuracy

1.5x10^8 (0.5 MF)

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How can a standardized inoculum be prepared?

Use 3-10 colonies of the same type in log phase

Indirect- inoculate broth and incubate until turbidity reached

Direct- emulsify colonies in broth to reach turbidity

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What are the characteristics of growth medium used for susceptibility testing?

Mueller Hinton broth/agar

pH- 7.2-7.4

Ca and Mg close to physiological

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Why must Ca and Mg concentrations be close to physiological conditions?

They affect the rate of aminoglycoside movement.

Low- false susceptibility

High- false resistance

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What is the quality control strain for growth medium?

P. aeruginosa

Used with an aminoglycoside antibiotic

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How can thymidine affect growth medium?

Can interfere with sulfonamide and trimethoprim results.

Can be used to bypass the PABA pathway these compounds inhibit.

Shows false resistance.

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What are the two forms of broth dilution susceptibility tests?

Macro

Micro

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How is the macro broth dilution test performed?

Serial doubling dilutions of antimicrobial

Standardized inoculum added to each

Incubated and inspected for growth

Lowest antimicrobial concentration with no growth is the minimal inhibitory concentration (MIC)

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What is the growth control for the macro method?

One tube gets no inoculum

If there’s growth, test is inaccurate

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How does the in vivo level of antimicrobial compare with the in vitro MIC?

In vivo should be 2-4x higher

Manufacturers determine what the in vivo level should be

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What is the minimal bactericidal concentration (MBC) and how is it determined?

Lowest antimicrobial concentration that kills 99.9% of bacteria.

MIC determined

Tubes without growth are subcultured

Plate with 99.9% lack of bacterial growth (death) is the MBC

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How is the micro broth dilution test performed?

Plates with micro tube wells contain antimicrobials

Each is inoculated

Incubated and examined for growth

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What are the controls for the micro method?

Sterility- not inoculated, should be no growth

Growth- inoculated, no antimicrobial, should have growth

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How can the antimicrobial in micro plates be stored?

Frozen (-20)

Lyophilized (4 or room temperature), require more inoculum to rehydrate

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What automated system can be used for the micro broth dilution method? Describe it.

Vitek

Identifies organisms and performs susceptibility testing

Inoculum is loaded into cassette which is loaded into the reader/incubator

Can be programmed to store data

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What are the advantages of automated micro testing?

Faster

More accurate

Automatic patient result transfer

Maldi-Tof can perform bacterial identification

Can sample mixed cultures

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What should be done to mixed cultures?

The inoculum should be subcultured on a plate to check purity.

Must be observed before results are released.

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How are agar dilution susceptibility tests performed?

Antimicrobials are diluted in agar

Inoculated with standardized inoculum

MIC determine by lowest concentration of antimicrobial that shows no growth in plates (susceptible)

How are agar plates inoculated for susceptibility testing?

Using Steer’s replicators

Fit multiple wells of standardized inoculum of different bacteria

Multiple probes transferred to each plate

What are the advantages of agar dilution testing?

Cheap

Test can be customized

Can be adapted for fastidious organisms

How is disc diffusion/Kirby Bauer testing performed?

Plate is inoculated with standardized broth (usually 0.5 MF) Antimicrobial discs are placed on the surface Antimicrobial diffuses out, forms a zone of inhibition where the concentration is great enough to stop bacterial growth The zones are measured and compared to the interpretive chart provided by CLSI

What phase are bacteria chosen for disc diffusion in?

Log phase Stationary/death- growth takes to long, larger zones, falsely sensitive

What factors affect antimicrobial disc diffusion and how?

Size of molecule- bigger diffuse slower Agar depth- thin gives falsely large zones Medium composition- stiff slows diffusion, falsely small zones Surface moisture pH Time between inoculation and disc application- >15min, falsely small zones Time between disc application and incubation- >15min, falsely large zones Broth concentration- >1.5x10^8CFU/mL, falsely small zones

What is the critical point?

The lowest antimicrobial concentration around the disc that inhibits growth. Corresponds to MIC.

What is a regression graph?

Prepared for each antimicrobial Zone size is plotted against MIC Regression line is drawn

How is the break point of an antimicrobial determined from a regression graph?

Draw a line that is at half in vivo MIC to the regression line Draw down to the zone size axis This value is the break point (lowest concentration that bacteria are susceptible to)

What are the incubation standards for disc diffusion?

35°C Ambient air (no extra CO2) 16-24hr

How are colonies selected for disc diffusion?

More than one colony of the same type Mixed cultures aren't used Purity checked by examining the sensitivity plate or subculturing the broth

What are the requirements for Mueller Hinton agar?

90-140mm Depth- 4-6mm Poured on level surface, flat bottom Stored in fridge (warmed to room temp before use) Dry surface

How are disc diffusion plates streaked?

Three directions Show confluent growth

What is the objective of clinical microbiology?

Bacterial identification Antimicrobial susceptibility

What situations require special zone size interpretations?

Colonies within zone- resistant subpop, check purity Swarming into zone- Proteus, ignore Inner ring- measure inner Fine veil of growth- sulfonamide or trimethoprim, ignore, measure 80% inhibition Sharp irregular zone- B-lactams, resistant, B-lactamase action

What are intermediate organisms?

Difficult to test and produce results of uncertain significance.

What are moderately susceptible organisms?

An infection that can be treated if higher dosages of the antimicrobial are used.

What bacteria can Kirby Bauer tests be used for?

Fast growers Non-fastidious Able to grow in ambient air Staphylococcus, enterococci, enteriobacteriaceae, nonfermentative G- rods

What are QC organisms used for?

To check the accuracy of susceptibility tests.

When are QC run for susceptibility tests?

When new discs/antimicrobial panels are received Whenever susceptibility tests are performed

What organisms are used for susceptibility test QC?

S. aureus, E. coli, P. aeruginosa Min set required

What is a major cause of susceptibility test inaccuracy?

Deterioration and loss of antimicrobial potency Especially B-lactam

How should antimicrobial discs be stored?

Less than 20°C Moisture free environment

How is MRSA susceptibility detected?

Directly prepare inoculum NaCl to broth and agar (enhance growth) Incubate at 30-35°C Disc diffusion- watch for trailing at zone edge

What is the oxacillin screen test? How is it performed?

Used for MRSA susceptibility MH agar with 6ug/mL oxacillin and 4% NaCl Spot inoculate Any growth- resistance

How is penicillin-resistant S. pneumoniae tested for susceptibility?

Inoculum streaked on MH agar enriched with blood 1ug oxacillin applied Incubated in CO2 Zones >20mm indicate susceptibility

How is Haemophilus susceptibility testing done?

Fastidious bacteria Modified MH agar, haemophilus test medium (HTM) Bovine heparin, yeast extract, NAD, thymidine phosphorylase

How is N. gonorrhoea susceptibility testing done?

Fastidious bacteria Special medium incubated in CO2 GC agar base enriched to allow growth

What is the epsilometer test?

A strip with predefined antimicrobial concentration gradient on one side and an MIC scale on the other side Applied to plate Creates elliptical inhibitory zone, MIC where the zone intersects with the scale

What if during an E-test two different levels are reached on either strip side?

The higher MIC is taken

What are the advantages of E-testing?

Can be used for bacteria that can't be tested traditionally Can use a medium that will support group

What do the results of an E-test indicate?

Low MIC- sensitive High MIC- resistant