Ritu's Biology Hub

Continued from Part 4

Factors Influencing Antimicrobial Drug Effectiveness

• Drug must be able to reach the site of infection
  • Various factors that control drug activity, stability and metabolism in vivo are important considerations during drug formulation. Mode of administration is essential factor. Penicillin G cannot be given orally because it gets degraded by stomach acid. Gentamicin and other aminoglycosides are not well absorbed from the intestinal tract and therefore, need to be given intramuscularly
  • Some agents may be simply excluded from the site of infection even if administered properly. Blood clots, necrotic tissues or biofilms hinder the penetrance of drug into the localized infection site. Alternatively, the agent could get absorbed by surrounding materials

• Pathogen must be susceptible to the drug
  • Bacteria in biofilms or abscesses may be replicating very slowly and thus, resistant to chemotherapy as most drugs act on actively growing bacteria
  • A pathogen actively growing may simply not be susceptible to the drug for the lack of cellular target. Example, penicillins and cephalosporins do not act on mycoplasmas due to the absence of cell wall. Hence, the drug should be chosen wisely by the practitioner taking various factors into account
  • Drug cocktails may be used to treat some infections such as use of Clavulanic acid (to inactivate penicillinase) and Ampicillin (to treat penicillin resistant bacteria).
  • Bacteriostatic drugs should not be given along with bactericidal drugs because the bacteriostatic agent will inhibit the growth of the pathogen while a bactericidal agent needs an actively growing and metabolically active cell to act upon. Hence, it will be rendered inactive

• The antimicrobial agent must exceed the MIC value of the pathogen in order to be effective
  • The concentration attained at the site of infection depends on the drug administered, route of administration, uptake speed and the rate at which the drug is eliminated from the body
  • A drug remains at high concentrations longer if its absorbed over an extended period and excreted slowly

Determination of Antimicrobial Effectiveness

This is absolutely essential for a proper therapy. Antimicrobial Susceptibility test (Antimicrobial Sensitivity test) allows us to determine which agents are most effective against pathogen and gives us an estimate of the therapeutic dose.

1. Dilution Susceptibility Tests

• Can be used to determine MIC and MLC
• Can be done in both agar and broth
• In broth dilution method, a series of tubes containing Mueller-Hinton (MH) broth containing antibiotic concentration from 0.1-128 µg/ml by 2 fold dilution are prepared
• A fixed volume of a microbial culture adjusted to 0.5 McFarland density is added to the tubes. A control, devoid of the antibiotic, is also maintained
• The lowest concentration of the antibiotic resulting in no growth after an incubation period of 24 hours at 37°C is the MIC. This is determined by assessing the turbidity in the tubes
• MLC is determined by subculturing the tubes showing no turbidity in fresh medium containing no antibiotic
• The lowest concentration of the antibiotic onwards which no growth occurs when transferred to a fresh medium lacking the antimicrobial agent is the MLC
• In the agar dilution method, solid MH agar plates containing increasing concentration (2 fold dilution) of an antibiotic are inoculated with a fixed volume of microbial culture whose density is adjusted to 0.5 McFarland density
• The plates are then incubated for 24 hours at 37°C and subsequently checked for growth. MIC and MLC is determined in the same way as above

2. Disk Diffusion Test

• Rapid test, hence, saves time and media
• Used for rapidly growing aerobic or facultative anaerobe pathogens like Staphylococcus and Pseudomonas
• When an antibiotic impregnated paper disk is placed on an agar previously inoculated with the test bacterium , the antibiotic diffuses rapidly radially outward through the medium producing an antibiotic concentration gradient
• The concentration is the greatest near the disk that inhibits growth of even minimally susceptible organisms. Resistant organisms grow right upto the disk
• As the distance increases from the disk, the concentration of the antimicrobial decreases and only more susceptible organisms are affected
• A clear zone around the disk implies that it inhibits the bacteria. Wider the zone, more susceptible is the pathogen
• Zone width is also affected by other factors such as initial concentration of the antibiotic, its solubility, diffusion rate through agar, thickness of the agar and the inoculum density
  • Greater the concentration of the drug, wider will be the zone of inhibition. Hence, all the test antibiotics should be more or less of the same concentration. Ideally, it should meet the standards.
  • More water-soluble the drug, greater diffusion through the agar
  • Drugs with higher diffusibility rate will diffuse greater through the agar than the ones with lower diffusibility rate. This also depends on the property of solubility of the drug in water
  • Thicker the agar, less diffusible will be the drug. So we shall get a false resistant result. Similarly, if the agar is too thin, the drug will diffuse more and we shall see a false susceptible result. Hence, this must be standardized.
  • If the inoculum is too dense, the organisms will grow closer to the disk giving a false resistant result and vice-versa
• The organism is first grown on a non-selective medium and suspension culture is made in a broth. The organism is allowed to grow in the medium by incubating the tube at 37°C for 24 hours.
• Density is adjusted to 0.5 McFarland density. A sterile cotton swab is dipped into the suspension and inoculated on the entire surface of MH agar. The agar should not be too wet.
• After drying the culture on the medium, antibiotic disks are placed on the medium either with a sterile forceps or a multiple applicator device.
• The plate is incubated at 37°C for 24 hours and checked for zones of inhibition which are measured in mm.
• Kirby Bauer test results are interpreted using a table that relates zone diameter to the degree of microbial resistance

3. Etest

• Convenient to use for anaerobic pathogens
• MH agar plate is streaked in 3 different directions with a sterile cotton swab dipped into a bacterial suspension culture
• Special strips impregnated with antibiotic in a graded manner are placed on the medium. They are labelled with MIC values
• After incubation, an elliptical zone of inhibition is observed. MIC value will be that value on the strip from which the zone of inhibition starts