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TESTING MICROBIAL SUSCEPTIBILITY TO

CHEMOTHERAPEUTIC AGENTS
I. Chemotherapeutic agents
Chemical substance used for treatment of infectious diseases caused by the proliferation of malignant
cells.

II. Antibiotics
Synthetic antibiotics
o Chemically-synthesized substances used to treat infectious disease
o Trimethoprim, exambutol, ciprofloxacin
o Antiviral agents
AZT, amantadine, Tamiflu are not considered antibiotics
Valciclovir (Valtrex), Acyclovir (Zovirax)
o Antifungal
Have selective toxicity for yeasts or other fungi but typically do not affect bacteria
o Antiparasitic
Anti-protozoal and anti-helminthic
Used for treatment of infections caused by parasites and worms
Semi-synthetic antibiotics
o Derivative of a natural antibiotic
o Molecular version produced by a microbe is subsequently modified by the chemist to achieve
desired properties
o Ampicillin, amoxicillin, doxycycline, rifampicin
Characteristics
o Spectrum of action
Range of bacteria or other microorganisms that is affected by a certain antibiotic
Broad spectrum
Effective against a wide range of gram positive and gram negative and possibly
some eukaryotes
Narrow spectrum
Effective mainly against gram positive OR gram negative bacteria
Limited spectrum
Effective against a single organism or disease
o Selective toxicity
The agent acts in some way that inhibits or kills the microbial pathogen but has little or no
toxic effect on the host
Cidal
o Killing
o Determined in vitro BY EXPOSING a standardized concentration of
organisms against a series of antimicrobial dilutions
o MINIMUM BACTERICIDAL CONCENTRATION
Lowest concentration that kills 99.9% of the population
Static
o Inhibitory
o Determined in vitro BY TESTING a standardized concentration of organisms
against a series of antimicrobial dilutions
o MINIMUM INHIBITORY CONCENTRATION
Lowest concentration that inhibits the growth of the organism
Lytic
o Lyse cells
Antibiotic synergism and antagonism
o SYNERGISM
Combination of two antibiotics that have enhanced bactericidal activity when tested
together compared with the activity of each antibiotic
o ANTAGONISM
Combination of antibiotics in which the activity of one antibiotic interferes with the activity
of the other
The sum of the activity is less than the activity of the most active individual drug
Microorganisms that produce antibiotics
o Molds
Produce Beta-lactam antibiotics
Penicillium
Cephalosporium
o Actinomycetes
Streptomyces species
Bacillus species
B. polymyxa
o Polymyxin
Bacillus subtilis
o bacitracin
Desirable properties of a clinically useful antibiotic
o Wide spectrum of activity with the ability to destroy or inhibit many different species of pathogenic
organisms
o Nontoxic to the host and without undesirable side effect
o Non allergenic to the host
o Not eliminate the normal flora of the host
o Not be able to reach the part of the human body
where the infection is occurring
o Inexpensive and easy to produce
o Chemically stable
Long shelf life
o Microbial resistance in uncommon and unlikely to
develop
Susceptibility to chemotherapeutic agents methods
o THINGS TO REMEMBER
The size of the inoculum should be
standardized
Turbidity of the log phase inoculum
should be adjusted to equal that of
the turbidity standard
o 0.5% McFarlands
0.05mL of 1.175%
Barium chloride and
9.95mL of Hydrogen
Sulfate
Between 1x10^8 and 2x10^8
CFU/mL of E. coli / P. aeruginosa / S. aureus
MUELLER-HINTON AGAR
Always used as test medium
Does not contain Para-aminobenzoic acid
o Inhibits the activity of sulphonamides being tested
Should not be older than three days from their day of preparation
o To ensure optimum hydration needed for the diffusion of the agents under
test
Cotton swab with inoculums is streaked on the surface of the MHA plates 3 times
o To allow the whole surface to be inoculated
Acceptable batch to batch reproducibility
Low in sulfonamide, trimethoprim, tetracycline inhibitors
Supports satisfactory growth of most non fastidious pathogens
Should be poured or be in a 4mm thick
pH should be 7.2-7.4
o Tube or agar dilution method
Determine the smallest amount of
chemotherapeutic agent required to inhibit
the growth of the organism in vitro
Decreasing concentrations of the
chemotherapeutic agent(s) are prepared in
twofold dilutions
Mueller-Hinton with cations
magnesium and calcium
Broth dilution susceptibility testing:
Antibiotics
o Methicilin
o Oxacillin
o Nafcillin
o Cephalosphorin
o Kirby-Bauer method
Paper disk agar diffusion method
Developed by Kirby Bauer in 1965
Correlate the sizes of the zone of inhibition
with the Minimum Inhibitory Concentration of
the drug or the microorganism under test
It is possible to determine whether the microorganism is resistant or susceptible to the
agent

RESULTS AND INTERPRETATION: