Introduction
Antimicrobial Chemotherapeutic Agents
Antimicrobial chemotherapy is the use of chemicals to inhibit or kill
microorganisms in or on the host. Chemotherapy is based on selective toxicity.
This means that the agent used must inhibit or kill the microorganism in
question without seriously harming the host.
In order to be selectively toxic, a chemotherapeutic agent must interact with
some microbial function or microbial structure that is either not present or is
substantially different from that of the host. For example, in treating infections
caused by prokaryotic bacteria, the agent may inhibit peptidoglycan synthesis
or alter bacterial (prokaryotic) ribosomes. Human cells do not contain
peptidoglycan and possess eukaryotic ribosomes. Therefore, the drug shows
little if any effect on the host (selective toxicity). Eukaryotic microorganisms, on
the other hand, have structures and functions more closely related to those of
the host. As a result, the variety of agents selectively effective against eukaryotic
microorganisms, such as fungi and protozoans, is small when compared to the
number available against prokaryotes. Also keep in mind that viruses are not
cells and, therefore, lack the structures and functions altered by antibiotics, so
antibiotics are not effective against viruses.
Based on their origin, there are 2 general classes of antimicrobial
chemotherapeutic agents:
- Antibiotics: substances produced as metabolic products of one microorganism,
which inhibit or kill other microorganisms.
- Antimicrobial chemotherapeutic chemicals: chemicals synthesized in the
laboratory, which can be used therapeutically on microorganisms.
Today the distinction between the 2 classes is not as clear, since many
antibiotics are extensively modified in the laboratory (semisynthetic) or even
synthesized without the help of microorganisms.
Most of the major groups of antibiotics were discovered prior to 1955, and
most antibiotic advances since then have come about by modifying the older
forms. In fact, only 3 major groups of microorganisms have yielded useful
antibiotics: the actinomycetes (filamentous, branching soil bacteria such as
Streptomyces), bacteria of the genus Bacillus, and the saprophytic molds Penicillium
and Cephalosporium.
To produce antibiotics, manufacturers inoculate large quantities of medium
with carefully selected strains of the appropriate species of antibiotic-producing
microorganism. After incubation, the drug is extracted from the medium and
purified. Its activity is standardized and it is put into a form suitable for administration.
Some antimicrobial agents are cidal in action: they kill microorganisms (e.g.,
penicillins, cephalosporins, streptomycin, neomycin). Others are static in action:
they inhibit microbial growth long enough for the body’s own defenses to
remove the organisms (e.g., tetracyclines, erythromycin, sulfonamides).
Antimicrobial agents also vary in their spectrum. Drugs that are effective
against a variety of both Gram-positive and Gram-negative bacteria are said to
be “Broad Spectrum” (e.g., tetracycline, streptomycin, cephalosporins, ampicillin,
sulfonamides). Those effective against just Gram-positive bacteria, just Gramnegative
bacteria, or only a few species are termed “narrow spectrum” (e.g.,
penicillin G, erythromycin, clindamycin, gentamycin).
If a choice is available, a narrow spectrum is preferable since it will cause
less destruction to the body’s normal flora. In fact, indiscriminate use of broadspectrum
antibiotics can lead to superinfection by opportunistic microorganisms,
such as Candida (yeast infections) and Clostridium difficile (antibiotic-associated
ulcerative colitis), when the body’s normal flora are destroyed. Other dangers
from indiscriminate use of antimicrobial chemotherapeutic agents include drug
toxicity, allergic reactions to the drug, and selection for resistant strains of
microorganisms.
Below are examples of commonly used antimicrobial chemotherapeutic
agents arranged according to their mode of action:
Antimicrobial agents that inhibit peptidoglycan synthesis
Inhibition of peptidoglycan synthesis in actively dividing bacteria results in
osmotic lysis. (A list of common antimicrobial chemotherapeutic agents listed by
both their generic and brand names and arranged by their mode of action.)
- Penicillins (produced by the mold Penicillium): There are several classes of
penicillins:
- Natural penicillins are highly effective against Gram-positive bacteria
(and very few Gram-negative bacteria) but are inactivated by the bacterial
enzyme penicillinase. Examples include penicillin G, F, X, K, O, and V.
- Semisynthetic penicillins are effective against Gram-positive bacteria but
are not inactivated by penicillinase. Examples include methicillin, dicloxacillin,
and nafcillin.
- Semisynthetic broad-spectrum penicillins are effective against a variety of
Gram-positive and Gram-negative bacteria but are inactivated by
penicillinase. Examples include ampicillin, carbenicillin, oxacillin, azlocillin,
mezlocillin, and piperacillin.
- Semisynthetic broad-spectrum penicillins combined with beta-lactamase
inhibitors such as clavulanic acid and sulbactam. Although the clavulanic
acid and sulbactam have no antimicrobial action of their own, they
inhibit penicillinase, thus protecting the penicillin from degradation.
Examples include amoxicillin plus clavulanic acid, ticarcillin plus clavulanic
acid, and ampicillin plus sulbactam.
- Cephalosporins (produced by the mold Cephalosporium): Cephalosporins are
effective against a variety of Gram-positive and Gram-negative bacteria
and are resistant to penicillinase (although some can be inactivated by
other beta-lactamase enzymes similar to penicillinase). Four “generations”
of cephalosporins have been developed over the years in an attempt to
counter bacterial resistance.
- First-generation cephalosporins include cephalothin, cephapirin, and
cephalexin.
- Second-generation cephalosporins include cefamandole, cefaclor, cefazolin,
cefuroxime, and cefoxitin.
- Third-generation cephalosporins include cefotaxime, cefsulodin, cefetamet,
cefixime, ceftriaxone, cefoperazone, ceftazidine, and moxalactam.
- Carbapenems: Carbapenems consist of a broad-spectrum beta-lactam antibiotic
to inhibit peptidoglycan synthesis combined with cilastatin sodium, an
agent that prevents degradation of the antibiotic in the kidneys. An example
is imipenem.
- Monobactems: Monobactems are broad-spectrum beta-lactam antibiotics
resistant to beta lactamase. An example is aztreonam.
- Carbacephem: A synthetic cephalosporins. An example is loracarbef.
- Vancomycin (produced by the bacterium Streptomyces): Vancomycin and
teichoplanin are glycopeptides that are effective against Gram-positive
bacteria.
- Bacitracin (produced by the bacterium Bacillus): Bacitracin is used topically
against Gram-positive bacteria.
Antimicrobial agents that alter the cytoplasmic membrane
Alteration of the cytoplasmic membrane of microorganisms results in leakage of
cellular materials. The following is a list of common antimicrobial chemotherapeutic
agents listed by both their generic and brand names and arranged
by their mode of action.
- Polymyxin B (produced by the bacterium Bacillus): Polymyxin B is used for
severe Pseudomonas infections.
- Amphotericin B (produced by the bacterium Streptomyces): Amphotericin B
is used for systemic fungal infections.
- Nystatin (produced by the bacterium Streptomyces): Nystatin is used mainly
for Candida yeast infections.
- Imidazoles (produced by the bacterium Streptomyces): The imidazoles are
antifungal antibiotics used for yeast infections, dermatophytic infections,
and systemic fungal infections. Examples include clotrimazole, miconazole,
ketoconazole, itraconazole, and fluconazole.
Antimicrobial agents that inhibit protein synthesis
The following is a list of common antimicrobial chemotherapeutic agents listed
by both their generic and brand names and arranged by their mode of action.
These agents prevent bacteria from synthesizing structural proteins and enzymes.
- Agents that block transcription (prevent the synthesis of mRNA off DNA).
- Rifampins (produced by the bacterium Streptomyces): Rifampins are
effective against some Gram-positive and Gram-negative bacteria and
Mycobacterium tuberculosis. They inhibit the enzyme RNA polymerase.
- Agents that block translation (alter bacterial ribosomes to prevent mRNA
from being translated into proteins).
- Agents such as the aminoglycosides (produced by the bacterium
Streptomyces) that bind irreversibly to the 30S ribosomal subunit and
prevent the 50S ribosomal subunit from attaching to the translation
initiation complex. Aminoglycosides also cause a misreading of the
mRNA. Examples include streptomycin, kanamycin, tobramycin, and amikacin. Most are effective against Gram-positive and Gram-negative
bacteria.
- Agents that bind reversibly to the 30S ribosomal subunit in such a way
that anticodons of charged tRNAs cannot align properly with the codons
of the mRNA. Examples include tetracycline, minocycline, and
doxycycline, produced by the bacterium Streptomyces. They are effective
against a variety of Gram-positive and Gram-negative bacteria.
- Agents that bind reversibly to the 50S ribosomal subunit and block
peptide bond formation during protein synthesis. Examples include
lincomycin and clindamycin, produced by the bacterium Streptomyces.
Most are used against Gram-positive bacteria.
- Agents that bind reversibly to the 50S ribosomal subunit and block
translation by inhibiting elongation of the protein by the enzyme
peptidyltransferase that forms peptide bonds between the amino acids,
by preventing the ribosome from translocating down the mRNA, or
both. Macrolides such as erythromycin, roxithromycin, clarithromycin,
and azithromycin are examples and are used against Gram-positive
bacteria and some Gram-negative bacteria.
- The oxazolidinones (linezolid) bind to the 50S ribosomal subunit and
appear to interfere with the initiation of translation.
- The streptogramins (a combination of quinupristin and dalfopristin)
bind to different sites on the 50S ribosomal subunit and work
synergistically to inhibit translocation.
Antimicrobial agents that interfere with DNA synthesis
The following is a list of common antimicrobial chemotherapeutic agents listed
by both their generic and brand names and arranged by their mode of action.
- Fluoroquinolones (synthetic chemicals): The fluoroquinolones inhibit one or
more of a group of enzymes called topoisomerase, enzymes needed for
bacterial nucleic acid synthesis. For example, DNA gyrase (topoisomerase II)
breaks and rejoins the strands of bacterial DNA to relieve the stress of the
unwinding of DNA that occurs during DNA replication and transcription.
Fluoroquinolones are broad spectrum, and examples include norfloxacin,
ciprofloxacin, enoxacin, levofloxacin, and trovafloxacin.
- Sulfonamides and trimethoprim (synthetic chemicals): Cotrimoxazole is a
combination of sulfamethoxazole and trimethoprim. Both of these drugs
block enzymes in the bacteria pathway required for the synthesis of
tetrahydrofolic acid, a cofactor needed for bacteria to make the nucleotide
bases thymine, guanine, uracil, and adenine.
- Metronidazole is a drug that is activated by the microbial proteins flavodoxin
and feredoxin, found in microaerophilc and anaerobic bacteria and certain
protozoans. Once activated, the metronidazole puts nicks in the microbial
DNA strands.
Microbial Resistance to Antimicrobial Chemotherapeutic Agents
A common problem in antimicrobial chemotherapy is the development of resistant
strains of bacteria. Most bacteria become resistant to antimicrobial agents by
one or more of the following mechanisms:
- Producing enzymes which detoxify or inactivate the antibiotic, e.g.,
penicillinase and other beta-lactamases.
- Altering the target site in the bacterium to reduce or block binding of the
antibiotic, e.g., producing a slightly altered ribosomal subunit that still
functions but to which the drug can’t bind.
- Preventing transport of the antimicrobial agent into the bacterium, e.g.,
producing an altered cytoplasmic membrane or outer membrane.
- Developing an alternate metabolic pathway to bypass the metabolic step
being blocked by the antimicrobial agent, e.g., overcoming drugs that
resemble substrates and tie up bacterial enzymes.
- Increasing the production of a certain bacterial enzyme, e.g., overcoming
drugs that resemble substrates and tie up bacterial enzymes.
These changes in the bacterium that enable it to resist the antimicrobial
agent occur naturally as a result of mutation or genetic recombination of the
DNA in the nucleoid, or as a result of obtaining plasmids from other bacteria.
Exposure to the antimicrobial agent then selects for these resistant strains of
organism.
The spread of antibiotic resistance in pathogenic bacteria is due to both
direct and indirect selection. Direct selection refers to the selection of antibioticresistant
pathogens at the site of infection. Indirect selection is the selection of
antibiotic-resistant normal floras within an individual anytime an antibiotic is
given. At a later date, these resistant normal floras may transfer resistance genes
to pathogens that enter the body. In addition, these resistant normal flora may
be transmitted from person to person through such means as the fecal-oral route
or through respiratory secretions.
As an example, many Gram-negative bacteria possess R (resistance) plasmids
that have genes coding for multiple antibiotic resistance through the mechanisms
stated above, as well as transfer genes coding for a sex pilus. Such an organism
can conjugate with other bacteria and transfer an R plasmid to them. Escherichia
coli,
Proteus,
Serratia, Salmonella, Shigella, and Pseudomonas are examples of bacteria
that frequently have R plasmids. Because of the problem of antibiotic resistance,
antibiotic susceptibility testing is usually done in the clinical laboratory to
determine which antimicrobial chemotherapeutic agents will most likely be
effective on a particular strain of microorganism. This is discussed in the next
section.
To illustrate how plasmids carrying genes coding for antibiotic resistance
can be picked up by antibiotic-sensitive bacteria, in today’s lab we will use
plasmid DNA to transform an
Escherichia coli sensitive to the antibiotic ampicillin
into one that is resistant to the drug.
The
E. coli. will be rendered more “competent” to take up plasmid DNA
(pAMP), which contains a gene coding for ampicillin resistance, by treating
them with a solution of calcium chloride, cold incubation, and a brief heat
shock. They will then be plated on 2 types of media: Lauria-Bertani agar (LB)
and Lauria-Bertani agar with ampicillin (LB/amp). Only
E. coli. that have picked
up a plasmid coding for ampicillin resistance will be able to form colonies on
the LB/amp agar.
Antibiotic Susceptibility Testing
For some microorganisms, susceptibility to chemotherapeutic agents is
predictable. However, for many microorganisms (
Pseudomonas,
Staphylococcus
aureus, and Gram-negative enteric bacilli such as
Escherichia coli,
Serratia,
Proteus,
etc.) there is no reliable way of predicting which antimicrobial agent will be
effective in a given case. This is especially true with the emergence of many
antibiotic-resistant strains of bacteria. Because of this, antibiotic susceptibility
testing is often essential in order to determine which antimicrobial agent to use
against a specific strain of bacterium.
Several tests may be used to tell a physician which antimicrobial agent is
most likely to combat a specific pathogen:
Tube dilution tests
In this test, a series of culture tubes are prepared, each containing a liquid
medium and a different concentration of a chemotherapeutic agent. The tubes
are then inoculated with the test organism and incubated for 16–20 hours at
35°C. After incubation, the tubes are examined for turbidity (growth). The lowest
concentration of chemotherapeutic agent capable of preventing growth of the
test organism is the minimum inhibitory concentration (MIC).
Subculturing of tubes showing no turbidity into tubes containing medium,
but no chemotherapeutic agent, can determine the minimum bactericidal
concentration (MBC). MBC is the lowest concentration of the chemotherapeutic
agent that results in no growth (turbidity) of the subcultures. These tests, however,
are rather time-consuming and expensive to perform.
The agar diffusion test (Bauer-Kirby test)
A procedure commonly used in clinical labs to determine antimicrobial
susceptibility is the Bauer-Kirby disc diffusion method. In this test, the in vitro
response of bacteria to a standardized antibiotic-containing disc has been
correlated with the clinical response of patients given that drug.
In the development of this method, a single high-potency disc of each
chosen chemotherapeutic agent was used. Zones of growth inhibition
surrounding each type of disc were correlated with the minimum inhibitory
concentrations of each antimicrobial agent (as determined by the tube dilution
test). The MIC for each agent was then compared to the usually attained blood
level in the patient with adequate dosage. Categories of “Resistant,”
“Intermediate,” and “Sensitive” were then established.
The basic steps for the Bauer-Kirby method of antimicrobial susceptibility
testing are:
- Prepare a standard turbidity inoculum of the test bacterium so that a
certain density of bacteria will be put on the plate.
- Inoculate a 150-mm Mueller-Hinton agar plate with the standardized
inoculum, so as to cover the entire agar surface with bacteria.
- Place standardized antibiotic-containing discs on the plate.
- Incubate the plate at 35°C for 18–20 hours.
- Measure the diameter of any resulting zones of inhibition in millimeters
(mm).
- Determine if the bacterium is susceptible, moderately susceptible, intermediate,
or resistant to each antimicrobial agent.
The term intermediate generally means that the result is inconclusive for
that drug-organism combination. The term moderately susceptible is usually
applied to those situations where a drug may be used for infections in a
particular body site, e.g., cystitis, because the drug becomes highly concentrated
in the urine.
Automated tests
Computerized automated tests have been developed for antimicrobial
susceptibility testing. These tests measure the inhibitory effect of the antimicrobial
agents in a liquid medium by using light-scattering to determine growth of the
test organism. Results can be obtained within a few hours. Labs performing
very large numbers of susceptibility tests frequently use the automated methods,
but the equipment is quite expensive.
Procedures
Microbial Resistance to Antimicrobial Chemotherapeutic Agents
Materials
Plasmid DNA (pAMP) on ice, calcium chloride solution on ice, 2 sterile culture
tubes, 1 tube of LB broth, 2 plates of LB agar, 2 plates of LB agar with
ampicillin (LB/amp), sterile 1-mL transfer pipettes, sterile plastic inoculating
loops, bent glass rod, turntable, alcohol, beaker of ice, water bath at 42°C.
Organism
LB agar culture of
Escherichia coli
Microbial Resistance Procedure
- Label one LB agar plate “Transformed bacteria, positive control” and the
other LB agar plate “Wild-type bacteria, positive control.”
Label one LB/amp agar plate “Transformed bacteria, experiment” and the
other LB/amp agar plate “Wild-type bacteria, negative control.”
- Label one sterile culture tube “(+)AMP” and the other “(–)AMP.” Using a
sterile 1-mL transfer pipette, add 250 µL of ice cold calcium chloride to
each tube. Place both tubes on ice.
Using a sterile plastic inoculating loop, transfer 1–2 large colonies of E. coli. into the (+)AMP tube and vigorously tap against the wall of the tube to
dislodge all the bacteria. Immediately suspend the cells by repeatedly
pipetting in and out with a sterile transfer pipette until no visible clumps
of bacteria remain. Return the tube to the ice.
- Repeat step 3, this time using the (–)AMP tube and return to the ice.
- Using a sterile plastic inoculating loop, add 1 loopful of pDNA (plasmid
DNA) solution to the (+)AMP tube and swish the loop to mix the DNA.
Return to the ice.
- Incubate both tubes on ice for 15 minutes.
- After 15 minutes, “heat-shock” both tube of bacteria by immersing them in
a 42°C water bath for 90 seconds. Return both tubes to the ice for 1 minute
or more.
- Using a sterile 1-mL transfer pipette, add 250 µL of LB broth to each tube.
Tap tubes with your fingers to mix. Set tubes in a test tube rack at room
temperature.
- Using a sterile 1-mL transfer pipette, add 100 mL of E. coli. suspension from
the (–)AMP tube onto the LB/amp agar plate labeled “Wild-type bacteria,
negative control.” Add another 100 L of E. coli. from the (–)AMP to the LB
agar plate labeled “Wild-type bacteria, positive control.”
- Using a bent glass rod dipped in alcohol and flamed, spread the bacteria
thoroughly over both agar plates. Make sure you reflame the glass rod
between plates.
- Using a sterile 1-mL transfer pipette, add 100 mL of E. coli. suspension from
the (+)AMP tube onto the LB/amp agar plate labeled “Transformed bacteria,
experiment.” Add another 100 L of E. coli. from the (+)AMP to the LB agar
plate labeled “Transformed bacteria, positive control.”
- Immediately spread as in step 10.
- Incubate all plates at 37°C.
Antibiotic Susceptibility Testing
Materials
- 150-mm Mueller-Hinton agar plates (3)
- Sterile swabs (3)
- An antibiotic disc dispenser containing discs of antibiotics commonly
effective against Gram-positive bacteria, and 1 containing discs of antibiotics
commonly effective against Gram-negative bacteria.
Organisms
- Trypticase soy broth cultures of Staphylococcus aureus (Gram-positive)
- Escherichia coli (Gram-negative), and Pseudomonas aeruginosa (Gram-negative)
Antibiotic Susceptibility Testing Procedure
- Take 3 Mueller-Hinton agar plates. Label one S. aureus, one E. coli., and
one P. aeruginosa.
- Using your wax marker, divide each plate into thirds to guide your
streaking.
- Dip a sterile swab into the previously standardized tube of S. aureus.
Squeeze the swab against the inner wall of the tube to remove excess
liquid.
- Streak the swab perpendicular to each of the 3 lines drawn on the plate,
overlapping the streaks to assure complete coverage of the entire agar
surface with inoculum.
- Repeat steps 3 and 4 for the E. coli. and P. aeruginosa plates.
- Using the appropriate antibiotic disc dispenser, place Gram-positive
antibiotic-containing discs on the plate of S. aureus and Gram-negative
antibiotic-containing discs on the plates of E. coli. and P. aeruginosa.
- Make sure that one of each of the antibiotic-containing discs in the dispenser
is on the plate, and touch each disc lightly with sterile forceps to make
sure it adheres to the agar surface.
- Incubate the 3 plates upside-down at 37°C until the next lab period.
- Using a metric ruler, measure the diameter of the zone of inhibition around
each disc on each plate in mm by placing the ruler on the bottom of the
plate.
- Determine whether each organism is susceptible, moderately susceptible,
intermediate, or resistant to each chemotherapeutic agent using the
standardized table, and record your results.
|
TABLE 1 Zone Size Interpretive Chart for Bauer-Kirby Test |
Result
Microbial Resistance to Antimicrobial Chemotherapeutic Agents
Count the number of colonies on each plate. If the growth is too dense to count
individual colonies, record “lawn” (bacteria cover nearly the entire agar surface).
Antibiotic Susceptibility Testing: Bauer-Kirby Method
Interpret the results following steps 9 and 10 of the procedure and record your
results.
Performance Objectives
Antimicrobial Chemotherapeutic Agents
- Define the following: antibiotic, antimicrobial chemotherapeutic chemical,
narrow-spectrum antibiotic, broad-spectrum antibiotic.
- Discuss the meaning of selective toxicity in terms of antimicrobial
chemotherapy.
- List 4 genera of microorganisms that produce useful antibiotics.
- Describe 4 different major modes of action of antimicrobial chemotherapeutic
chemicals, and name 3 examples of drugs fitting each mode of action.
Microbial Resistance to Antimicrobial Agents
Discussion
- Describe 5 mechanisms by which microorganisms may resist antimicrobial
chemotherapeutic agents.
- Briefly describe R plasmids and name 4 bacteria that commonly possess
these plasmids.
Results
Interpret the results of the
Escherichia coli plasmid transformation experiment.
Antibiotic Susceptibility Testing
Discussion
- Explain why antimicrobial susceptibility testing is often essential in choosing
the proper chemotherapeutic agent for use in treating an infection.
- Define MIC.
Results
Interpret the results of a Bauer-Kirby antimicrobial susceptibility test when
given a Mueller-Hinton agar plate, a metric ruler, and a standardized zone-size
interpretation table.