Antimicrobial Agents and Antimicrobial Susceptibility Testing

• 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)

• Dilutions of the antimicrobial agents are prepared in broth
• Assays are generally performed in microtiter plates

• Dilutions of the antimicrobial agents are prepared in agar
• Bacteria are inoculated onto the agar plates

The lowest concentration of an antimicrobial agent that kills at least 99.9% of the bacteria in the original inoculum

Kirby-Bauer sensitivity test

• 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

• Haemophilus test medium
• Mueller-Hinton base supplemented with hematin, NAD, and yeast extract

Bacterial inoculum (10⁸ colony forming units/mL)

• 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)

• Oxacillin or Nafcillin
• (Methicillin is no longer available in the US)

Cefoxitin

• Disk diffusion:  <=19 mm Resistant and >=20 mm Sensitive
• Broth dilution tests: <2 microgram/mL Sensitive and >4 microgram/mL Resistant

Etest (AB Biodisk) provides quantitative antimicrobial susceptibility testing results

• 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

An enzyme that confers resistance to penicillin and some of the semisynthetic penicillins (i.e. ampicillin)

Enterobacteriaceae

• Extended-spectrum beta-lactamase
• These enzymes inactivate the extended spectrum cephalosporins (i.e. ceftriaxone and cefotaxime)

• Extended-spectrum beta-lactamase (ESBL)
• These enzymes inactivate the extended spectrum cephalosporins (i.e. ceftriaxone and cefotaxime)

• 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

Inhibit cell wall synthesis

• Penicillins
• Cephalosporins
• Monobactams
• Carbapenems

• Narrow spectrum (1st generation)
• Expanded spectrum (2nd gen)
• Broad spectrum (3rd gen)
• Extended spectrum (4th gen)

• 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

• Clavulanic acid
• Sulbactam
• Tazobactam

• 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

• Gentamicin
• Tobramycin
• Netilmicin

• Tobramycin is a bactericidal
• Others are bacteristatic

• Inhibit protein synthesis at the 30S ribosomal subunit
• Active against gram-positive, gram-negative, Mycoplasma, and Chlamydia
• Increased resistance has limited their use

• Doxycycline
• Minocycline

• 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)

Inhibit protein synthesis

• Erythromycin
• Clarithromycin

Inhibit folic acid synthesis by forming nonfunctional analogs of folic acid

Inhibit cell wall formation by inhibiting peptidoglycan synthesis

Vancomycin

• Enterococcus
• Staph aureus

• Inhibit DNA activity by inactivating DNA gyrase
• Newer agents are known as fluoroquinolones

• Ciprofloxacin
• Levofloxacin

• Disrupt plasma membranes
• Used to treat infections caused by gram-negative bacteria

• Polymixin B
• Polymixin E

• Inhibits bacterial enzymes
• Used to treat UTIs

• Molecule produced my microorganism that inhibits the growth of other microorganisms
• Antibiotics can also be synthetic

• Kills microorganisms
• (i.e. bactericidal compound kills bacteria)

• Inhibits the growth of microorganisms
• (i.e. bacteristatic compound inhibits bacterial growth)

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

Limitied range of action

Active against a wide range of bacteria