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How does Dilution Antimicrobial testing work?
- Bacteria are exposed to different concentrations of antimicrobial agents
- The smallest concentration that inhibits growth of bacteria is recorded - this value is the minimal inhibitory concentration (MIC)
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How are Broth antimicrobial dilutions performed?
- Dilutions of the antimicrobial agents are prepared in broth
- Assays are generally performed in microtiter plates
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How are Agar Antimicrobial dilutions performed?
- Dilutions of the antimicrobial agents are prepared in agar
- Bacteria are inoculated onto the agar plates
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Define the minimum bactericidal concentration (MBC) of an antimicrobial agent
The lowest concentration of an antimicrobial agent that kills at least 99.9% of the bacteria in the original inoculum
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What is Disk diffusion antimicrobial testing also referred to as?
Kirby-Bauer sensitivity test
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What agar is used for the Standardization of Disk diffusion?
- Mueller-Hinton agar (MHA) - 4 mm thick in Petri dish at a pH 7.2-7.4
- Fastidious organisms (like S. pneumo) will use MHA with 5% sheep red blood cells
- For Haemophilus influenzae - Haemophilus test medium (HTM) is used
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What does HTM stand for and what nutrients are in it?
- Haemophilus test medium
- Mueller-Hinton base supplemented with hematin, NAD, and yeast extract
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What is equal to a McFarland #0.5 turbidity standard?
Bacterial inoculum (108 colony forming units/mL)
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How are disk diffusion rated for sensitivity?
- After incubation, the diameters of the zones of inhibition are measured
- Zone sizes are compared to standard interpretation charts
- Results are reported as sensitive (S), intermediate (I), or resistant (R)
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What is MRSA actually resistant to?
- Oxacillin or Nafcillin
- (Methicillin is no longer available in the US)
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When dealing with MRSA, what is a powerful inducer of oxacillin resistance?
Cefoxitin
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What are the zones and results for Oxacillin resistance for MRSA?
- Disk diffusion: <=19 mm Resistant and >=20 mm Sensitive
- Broth dilution tests: <2 microgram/mL Sensitive and >4 microgram/mL Resistant
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What test is done for Gradient diffusion?
Etest (AB Biodisk) provides quantitative antimicrobial susceptibility testing results
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What is the procedure for the Gradient diffusion Etest antimicrobial testing?
- Bacterial suspension equal to a McFarland #0.5 turbidity standard is prepared
- Bacteria are lawned onto Mueller-Hinton agar plate and the Etest strips are placed on top of the agar - each strip contains a different antimicrobial agent
- After incubation, the bacteria produce and elliptical zone of inhibition around the strip where the zone of inhibition crosses the strip
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What does beta-lactamase do?
An enzyme that confers resistance to penicillin and some of the semisynthetic penicillins (i.e. ampicillin)
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What organisms produce extended-spectrum beta-lactamase (ESBL)?
Enterobacteriaceae
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What is ESBL and what does it do?
- Extended-spectrum beta-lactamase
- These enzymes inactivate the extended spectrum cephalosporins (i.e. ceftriaxone and cefotaxime)
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What are the organisms Enterobacteriaceae capable of producing?
- Extended-spectrum beta-lactamase (ESBL)
- These enzymes inactivate the extended spectrum cephalosporins (i.e. ceftriaxone and cefotaxime)
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What is the D-zone test used for?
- Used to detect the presence of inducible clindamycin resistance by erythromycin
- A plate is inoculated for disk diffusion assay with erythromycin and clindamycin disk
- After incubation, the plate is examined for a flattening of the zone of inhibition around the clindamycin disk, resembling the letter D - indicating the presence of inducible resistance to clindamycin
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How do Beta-lactam antibiotics work?
Inhibit cell wall synthesis
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What are some examples of Beta-lactams?
- Penicillins
- Cephalosporins
- Monobactams
- Carbapenems
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How are the class of cephalosporins categorized?
- Narrow spectrum (1st generation)
- Expanded spectrum (2nd gen)
- Broad spectrum (3rd gen)
- Extended spectrum (4th gen)
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How do Beta-lactamase inhibitors work?
- Bacteria can exhibit resistance to the beta-lactam antibiotics by producing and enzyme (beta-lactamase) that cleaves the beta-lactam ring - inactivating the antibiotic
- Beta-lactamase inhibitors can be given with a beta-lactam antibiotic to provide effective treatment
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What are examples of Beta-lactamase inhibitors?
- Clavulanic acid
- Sulbactam
- Tazobactam
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How do Aminoglycosides work?
- Inhibit protein synthesis at the 30S ribosomal subunit
- Active against gram-negative and gram-positive bacteria
- No activity against obligate anaerobes
- Because of potential toxicity, dosage should be monitored using peak and trough values in peripheral blood
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What are some examples of Aminoglycosides?
- Gentamicin
- Tobramycin
- Netilmicin
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How is Tobramycin classified, compared to the other Aminoglycosides?
- Tobramycin is a bactericidal
- Others are bacteristatic
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How do Tetracyclines work?
- Inhibit protein synthesis at the 30S ribosomal subunit
- Active against gram-positive, gram-negative, Mycoplasma, and Chlamydia
- Increased resistance has limited their use
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What are some examples of Tetracyclines?
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How do Chloramphenicols work?
- Inhibits protein synthesis by binding to the 50S ribosomal subunit
- Broad spectrum
- Used to treat serious gram-negative infections, such as meningitis
- Risk of bone marrow toxicity, aplastic anemia (bone marrow suppression)
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How do Macrolides work?
Inhibit protein synthesis
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What are some examples of Macrolides?
- Erythromycin
- Clarithromycin
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How do Sulfonamides work?
Inhibit folic acid synthesis by forming nonfunctional analogs of folic acid
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How do Glycopeptides work?
Inhibit cell wall formation by inhibiting peptidoglycan synthesis
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What is the only approved Glycopeptide for the U.S.?
Vancomycin
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What are some Vancomycin resistant species isolated?
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How do Quinolones work?
- Inhibit DNA activity by inactivating DNA gyrase
- Newer agents are known as fluoroquinolones
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What are some examples of Fluoroquinolones (Quinolones)?
- Ciprofloxacin
- Levofloxacin
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How do Polymyxins work?
- Disrupt plasma membranes
- Used to treat infections caused by gram-negative bacteria
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What are some examples of Polymyxins?
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How do Nitrofurantoins work?
- Inhibits bacterial enzymes
- Used to treat UTIs
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Define antibiotic
- Molecule produced my microorganism that inhibits the growth of other microorganisms
- Antibiotics can also be synthetic
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Define Cidal
- Kills microorganisms
- (i.e. bactericidal compound kills bacteria)
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Define Static
- Inhibits the growth of microorganisms
- (i.e. bacteristatic compound inhibits bacterial growth)
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Define Synergy
When 2 or more antimicrobials are used and the combined effect is greater than what would be expected for the simple additive effect of the agents
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What does Narrow-spectrum antimicrobial agent mean?
Limitied range of action
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What does Broad-spectrum antimicrobial agent mean?
Active against a wide range of bacteria
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