FINALS LAB BACTE

Antimicrobials

Compounds that kill or inhibit microorganisms.

Antibiotics

Usually of low molecular weight, produced by microorganisms that inhibit or kill other microorganisms.

Penicillin

Targets cell wall/B-lactamase, peptidoglycan synthesis—amino acid side chain

Vancomycin

Targets cell wall/Peptidoglycan synthesis

Bacitracin

Targets cell wall/Transport of peptidoglycan monomer

Isoniazid

Targets cell wall/Mycolic acid synthesis in Mycobacterium

Fluroquinolones

Targets DNA/Topoisomerase unwinding of DNA in DNA synthesis

Rifamycins

Targets RNA/RNA polymerase in RNA synthesis

Tetracyclines and Streptomycin

Targets protein synthesis/30S subunit of 70S ribosome

Chloramphenicol

Targets protein synthesis/50S subunit of 70S ribosome

Sulfa drugs

Targets structural analogue of para-amino benzoic acid (PABA)— inhibit enzyme linking pteridine to PABA in folic acid synthesis

Penicillin, Fluroquinolones, Tetracyclines, Streptomycin, Chloramphenicol, Sulfa drugs

Cells affected: G+ and G-. What are the antibiotics?

Vancomycin, Bacitracin

Cell affected: G+. What are the antibiotics?

Isoniazid

Cell affected: Mycobacterium tuberculosis. What is the antibiotic?

Rifamycins

Cells affected: G+; some G-. What is the antibiotic?

Antimicrobial Susceptibility Testing

Performed on bacteria and fungi isolated from clinical specimens to D determine which antimicrobial agents might be effective in treating : infections caused by these organisms

to determine whether the bacterial isolate is capable of expressing resistance to the antimicrobial agents selected for treatment

What is the primary goal of antimicrobial Susceptibility Testing?

disk diffusion or dilution (minimal inhibitory concentration [MIC])

Antimicrobial Susceptibility Testing is often performed by?

Clinical and Laboratory Standards Institute (CLSI)

Standards that describe the antimicrobial susceptibility testing methods are published and frequently updated by

National Committee for Clinical Laboratory Standards (NCCLS)

Clinical and Laboratory Standards Institute (CLSI) is formerly known as?

Bacterial inoculum size, Growth medium, Incubation atmosphere, Incubation temperature, Incubation duration, Antimicrobial concentrations

The standardized components of AST include:

Mueller-Hinton base

Growth medium is typically a?

pH, Cation concentration, Blood and serum supplements, Thymidine content

Growth medium is composed of?

Inoculum, Media, Incubation

Primary Variables That Must Be Controlled in Performance of Routine Disk Diffusion and Broth Microdilution Minimal Inhibitory Concentration Tests

Formulation, Ca^{2+}, Mg^{2+} content, Thymidine content, pH, Agar depth (disk diffusion)

Media variable is composed of?

Atmosphere, Temperature, Length

Incubation variable is composed of?

1.5 x 10^8 CFU/mL

Standard disk diffusion

5 x 10^5 CFU/mL

Standard broth microdilution

Mueller-Hinton

Standard Media formulation in Performance of Routine Disk Diffusion and Broth Microdilution Minimal Inhibitory Concentration Tests

25 mg/L Ca^{2+}, 12.5 mg/L Mg^{2+}

Standard Ca^{2+}, Mg^{2+} content

Minimal or absent

Standard Thymidine content

7.2-7.4

Standard pH

3-5mm

Standard Agar depth (disk diffusion)

Humidified ambient air

Standard Atmosphere

35 degrees C

Standard Temperature

16-18 h

Standard Length for Disk diffusion

16-20 h

Standard Length for Broth microdilution:

24 h

Standard Length for staphylococci with oxacillin and vancomycin and for enterococci with vancomycin and gentamicin HLAR;

24 h

Length of time that is sometimes needed for fastidious bacteria

48 h

Standard Length for enterococci with streptomycin HLAR

0.5 for disk diffusion

Use adequate McFarland turbidity standard

>1 day old

When preparing direct suspensions (without incubation), do not use growth from plates

house or purchase

Prepare Mueller-Hinton in _________from reliable source

Increased concentrations

Calcium and magnesium content that may result in decreased activity of aminoglycosides against Pseudomonas aeruginosa

Increased concentrations

Calcium and magnesium content that may result in decreased activity of aminoglycosides against Pseudomonas aeruginosa and decreased activity of tetracyclines against all organisms (decreased concentrations have opposite effect)

Excessive concentrations

Thymidine content that can result in false resistance to sulfonamides and trimethoprim

Decreased pH

can lead to decreased activity of aminoglycosides, erythromycin, and clindamycin and increased activity of tetracyclines

Increased pH

can lead to increased activity of aminoglycosides, erythromycin, and clindamycin and decreased activity of tetracyclines

(<3 mm, >5 mm)

Possibility for false susceptibility if Agar depth is______ or false resistance if it is ______

CO2 incubation

this can decrease pH, which can lead to decreased activity of aminoglycosides, erythromycin, and clindamycin and increased activity of tetracyclines

(>35 degrees C)

Some MRSA may go undetected if the temperature is

(<24 h)

Some MRSA may go undetected if the length is

(<24 hr)

Some vancomycin-resistant enterococci may go undetected if the length is ______with disk diffusion

(<24 h)

Some HLAR (gentamicin) enterococci may go undetected if the length is _______ (broth microdilution)

(<48 h)

Some HLAR (streptomycin) enterococci may go undetected if the length is_____(broth microdilution)

probable cause, predicted

Antimicrobial susceptibility testing should be performed on a bacterial isolate from a clinical specimen if the isolate is determined to be a ____________of the patient's infection and the susceptibility of the isolate to particular antimicrobials cannot be reliably ________based on previous experience with the bacteria at a specific health care facility.

Susceptibility testing of isolates

provide information on decreases in the susceptibility of bacteria to antimicrobials

Body site, Presence of other organisms and quality of the specimen, Host's status

Factors to Consider When Determining Whether Testing Is Warranted

Disc Storage, Inoculum Preparation and Use of McFarland Standards

Traditional Antimicrobial Susceptibility Test Methods

Inoculum Preparation

McFarland Turbidity Standards

Inoculum Standardization

Inoculum Preparation and Use of McFarland Standards is composed of

−20 degrees C or below, non–frost-free freezer

What is the recommended long-term storage condition for antimicrobial disks?

2 - 8 degrees C, atleast 1 week in a refrigerator

How should a working supply of antimicrobial disks be stored?

Tightly sealed with desiccant

How should antimicrobial disks be stored within the container?

Warm to room temperature to prevent condensation

What must be done before opening the disk container?

Inoculum Preparation

one of the most critical steps in susceptibility testing

Suspend 4–5 colonies in broth, grow to log phase

How is the inoculum prepared from colonies?

suspending colonies grown overnight on an agar plate directly in broth or saline.

Inoculum preparation can also be prepared directly by?

For bacteria that grow unpredictably in broth.

When is direct suspension preparation preferred?

It doesn’t rely on broth growth

Why is the use of fresh 16–24-hour colonies in direct inoculum suspension preparation is imperative?

Compare turbidity to McFarland standard

How is inoculum size standardized?

By comparing suspension turbidity with a turbidity standard

How is standard inoculum size ensured?

Too few: false-susceptible; too many: false-resistant

What can happen if too few or too many bacteria are used in susceptibility testing?

By mixing 1% H2SO4 and 1.175% BaCl2

How are McFarland standards prepared?

99.5 mL of 1% H2SO4 + 0.5 mL of 1.175% BaCl2

How is McFarland 0.5 standard prepared?

vortexed thoroughly

What is done to the inoculated broth or suspension before comparing to McFarland standard?

against a white card with horizontal black lines under good lighting

How is the suspension compared to the McFarland 0.5 standard?

Diffusion method and dilution method

Methods of Antimicrobial Susceptibility Testing

Kirby-Bauer Diffusion Method (most : common), Agar Cup Diffusion Method, Agar Cylinder Diffusion Methods, Epsilometer/Gradient Diffusion Method

Diffusion method includes

Macrobroth Method/Tube Dilution Method, Microtube Dilution Method

Dilution method includes

Agar Diffusion Method/Disk Diffusion Method

Kirby-Bauer Method is also known as

Kirby-Bauer Method

-used to determine the sensitivity or resistance of a bacterium to an antimicrobial.

Mueller-Hinton Agar, 16–20 h

Kirby-Bauer Method method uses ___________ and requires ______ of incubation to measure zones of inhibition around antibiotic disks for susceptibility testing.

Preparation of pure inoculum, Standardize pure inoculum (using 0.5 McFarland Standard), Streak the pure inoculum into the medium (MHA), Apply antibiotic discs, Incubate, Measure the zone of inhibition

Kirby-Bauer Method step by step procedure

Mueller-Hinton Broth, Trypticase Soy Broth, Sterile Distilled Water, Natural Saline Solution, Brain Heart Infusion Broth

Preparation of pure inoculum, using any of the following:

add colonies, incubate

If standard more turbid than inoculum - _______ to inoculum or ______inoculum

add distilled water

If inoculum more turbid than standard - ____________ to inoculum

sterile cotton swab

In streaking, use a ___________ and streak with no space in between

Using forceps aseptically

How are antibiotic discs applied in the Kirby-Bauer method?

At least 15 mm apart

How far apart should antibiotic discs be spaced in the Kirby-Bauer method?

35 degrees C for 16–20 hours

What is the standard incubation condition in the Kirby-Bauer method?

Using a ruler or microcaliper

How is the zone of inhibition measured in the Kirby-Bauer method?

mm

What unit is used to measure the zone of inhibition?

Susceptible (S)

Indicates that the antimicrobial agent in question may be an appropriate choice for treating the infection caused by the organism. Bacterial resistance is absent or at a clinically insignificant level.

Intermediate (I)

• The potential utility of the antimicrobial agent in body sites where it may be concentrated (e.g., the urinary tract) or if high concentrations of the drug are used.

• Possible effectiveness of the antimicrobial agent against the isolate, but possibly less so than against a susceptible isolate.

• Use as an interpretive safety margin to prevent relatively small changes in test results from leading to major swings in interpretive category (e.g., resistant to susceptible or vice versa)

Resistant (R)

Indicates that the antimicrobial agent in question may not be an appropriate choice for treatment, either because the organism is not inhibited with serum-achievable levels of the drug or because the test result highly correlates with a resistance mechanism that indicates questionable successful treatment.

Dilution Susceptibility Testing Methods

• used to determine the MIC

• Various concentrations of an antimicrobial agent are added to broth or agar media.

nonsusceptible, susceptible, intermediate, or resistant

In Dilution Susceptibility Testing Methods, organism is interpreted as _____________________________

Antimicrobial Stock Solutions, Broth Macrodilution (Tube Dilution) Tests, Broth Microdilution Tests, Agar Dilution Tests

Dilution Susceptibility Testing Methods includes:

reference standard antimicrobial powders

Antimicrobial Stock Solutions must be prepared from __________________, not from the pharmaceutical preparations administered to patients