MCB 403

Concept of growth and death in microorganisms

Microbial growth
Growth is the orderly increase in the sum of all the components of an organism. Thus, the
increase in size that results when a cell takes up water or deposits lipid or polysaccharide is not
true growth. Cell multiplication is a consequence of growth; in unicellular organisms, growth
leads to an increase in the number of individuals making up a population or culture.
Microorganisms, such as bacteria, grow and reproduce through a process of cell division, where
a single cell divides into two daughter cells. These daughter cells then continue to grow and
divide, leading to an exponential increase in the number of cells in the population. Factors such
as nutrient availability, temperature, pH, and Moisture can all influence microbial growth and the
rate of cell division.

Bacterial Division
Most prokaryotes reproduce by binary fission, although some prokaryotes reproduce by budding,
fragmentation, and other means. Binary fission is a relatively simple type of cell division. During
binary fission, the cell elongates, replicates its chromosome, and separates the newly formed
DNA molecules so there is one chromosome in each half of the cell. Finally, a septum (or cross
wall) is formed at mid-cell, dividing the parent cell into two progeny cells, each having its own
chromosome and a complement of other cellular constituents. The time taken for a bacterial cell
to double is called generation time. The generation time varies among different species of
bacteria based on the environmental conditions they grow in. Clostridium perfringens is the
fastest growing bacteria that have a generation time of 10 minutes while Mycobacterium
tuberculosis is one of the slowest growing bacteria, taking about 16 to 20 hours to double.

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Figure 1. Binary Fission

Growth Curve
Binary fission and other cell division processes bring about an increase in the number of cells in
a population. Population growth is studied by analyzing the growth curve of a microbial culture.
When microorganisms are cultivated in liquid medium, they usually are grown in a batch culture
or closed system (i.e., they are incubated in a closed culture vessel with a single batch of
medium). Because no fresh medium is provided during incubation, nutrient concentrations
decline and concentrations of wastes increase. The growth of microorganisms reproducing by
binary fission can be plotted as the logarithm of the number of viable cells versus the incubation
time. The resulting curve has four distinct phases.

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Figure 2. Microbial Growth Curve in a Closed System

Lag Phase

The lag phase is the first phase of bacterial growth, during which the bacteria are adapting to
their new environment and are not yet dividing. During the lag phase, bacteria are actively
synthesizing new proteins, enzymes, and other cellular components needed for growth, as well as
repairing any damage caused by stress or changes in environmental conditions. The length of the
lag phase can vary depending on the bacterial species and the environmental conditions.

Exponential Phase
The second phase of the growth curve is the logarithmic growth phase (also known as the log
phase or exponential growth phase). In the log phase, the bacteria multiply so rapidly that the
number of organisms doubles with each generation time (i.e., the number of bacteria increases
exponentially). Growth rate is the greatest during the log phase. The log phase is always brief,
unless the rapidly dividing culture is maintained by constant addition of nutrients and frequent
removal of waste products. When plotted on logarithmic graph paper, the log phase appears as a
steeply sloped straight line.

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Stationary phase
As the nutrients in the liquid medium are used up and the concentration of toxic waste products
from the metabolizing bacteria build up, the rate of division slows, such that the number of
bacteria that are dividing equals the number that are dying. The result is the stationary phase. It is
during this phase that the culture is at its greatest population density. The production of
antibiotics such as penicillin and streptomycin, as well as certain enzymes, typically occurs
during the stationary phase of bacterial growth. During this phase, the bacteria may produce
antibiotics as a means of competing with other microorganisms for limited resources.

Death phase/Decline phase

As overcrowding occurs, the concentration of toxic waste products continues to increase and the
nutrient supply decreases. The microorganisms then die at a rapid rate; this is the death phase or
decline phase. The culture may die completely, or a few microorganisms may continue to survive
for months. If the bacterial species is a sporeformer, it will produce spores to survive beyond this
phase. When cells are observed in old cultures of bacteria in the death phase, some of them look
different from healthy organisms seen in the log phase. As a result of unfavorable conditions,
morphologic changes in the cells may appear.

Measurement of Microbial Growth

There are many ways to measure microbial growth to determine growth rates and generation
times. Either population number or mass may be followed because growth leads to increases in
both. Microbiologists sometimes need to know how many bacteria are present in a particular
liquid at any given time (e.g., to determine the degree of bacterial contamination in drinking
water, milk, and other foods). The microbiologist may (a) determine the total number of bacterial
cells in the liquid (the total number would include both viable and dead cells) or (b) determine
the number of viable (living) cells.

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Measurement of Cell Numbers

Several plating methods can be used to determine the number of viable microbes in a sample.
These are referred to as viable counting methods because they count only those cells that are
alive and able to reproduce. Two commonly used procedures are the spread-plate technique and
the pour-plate technique. For viable counting methods, a 1-mL volume is removed from a
bacterial suspension and serially diluted 10-fold followed by plating 0.1-mL aliquots on an agar
medium. Each single invisible bacterium (or clump of bacteria) will grow into a visible colony
that can be counted. For statistical purposes, plates containing between 30 and 300 colonies give
the most accurate data. Using this method, dead bacteria within the suspension do not contribute
to the final bacterial count.

Figure 3. Spread-Plate Technique

Measurement of Cell Mass

Increases in the total cell mass, as well as in cell numbers, accompany population growth.
Biomass can be measured directly by determining the dry weight of a microbial culture. Cells
growing in liquid medium are collected by centrifugation, washed, dried, and weighed. This
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method is time-consuming, however, and not very sensitive. Because bacteria weigh so little, it
may be necessary to centrifuge several hundred milliliters of culture to collect a sufficient
quantity.
Spectrophotometry can also be used to measure cell mass. In a spectrophotometer, a beam of
light is passed through the liquid. When no bacteria are present in the liquid, the liquid is clear,
and a large amount of light passes through. As bacteria increase in number, the liquid becomes
turbid (cloudy), and less light passes through. Turbidity increases (i.e., the solution becomes
more cloudy) as the number of organisms increases; therefore, the amount of transmitted light
decreases as the bacteria increase in number.

Chemistry of Synthetic Chemotherapeutic Agents and Antibiotics

For thousands of years, people have been discovering and using herbs and chemicals to cure
infectious diseases. Native witch doctors in Central and South America long ago discovered that
the herb, ipecac, aided in the treatment of dysentery, and that a quinine extract of cinchona bark
was effective in treating malaria. During the 16th and 17th centuries, the alchemists of Europe
searched for ways to cure smallpox, syphilis, and many other diseases that were rampant during
that period of history. Many of the mercury and arsenic chemicals that were used frequently
caused more damage to the patient than to the pathogen.

The chemicals (drugs) used to treat diseases are referred to as chemotherapeutic agents. By
definition, a chemotherapeutic agent is any drug used to treat any condition or disease. The
chemotherapeutic agents used to treat infectious diseases are collectively referred to as
antimicrobial agents. Thus, an antimicrobial agent is any chemical (drug) used to treat an
infectious disease, either by inhibiting or killing pathogens in vivo. Drugs used to treat bacterial
diseases are called antibacterial agents, whereas those used to treat fungal diseases are called
antifungal agents. Drugs used to treat protozoal diseases are called antiprotozoal agents, and
those used to treat viral diseases are called antiviral agents.

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 Characteristics of an ideal antimicrobial agent

 Selective toxicity: The aim of antimicrobial therapy is to kill or inhibit the infecting
organism without damaging the host; this is known as selective toxicity
 Should cause no allergic reaction in the host
 Should be stable when stored in solid or liquid form
 Should remain in specific tissues in the body long enough to be effective
 Should kill the pathogens before they mutate and become resistant to it.
 Should be active at low concentration
 Should be inexpensive and easy to produce
 Should not eliminate the normal flora of the host
The key characteristics of an ideal antimicrobial agent are: selective toxicity, potency, rapid
onset, stability, low toxicity, ease of use, and low resistance potential. Unfortunately, most
antimicrobial agents have some side effects, produce allergic reactions, or permit development of
resistant mutant pathogens.

Classifications of Antimicrobial Drugs

Antimicrobial drugs are classified in many ways:
(a) Based on the mechanisms of action of antimicrobial agents
The five most common mechanisms of action of antimicrobial agents are as follows:
(i) Inhibition of cell wall synthesis. Examples include: Penicillin, Cephalosporins,
Carbapenems

(ii) Damage to cell membranes. Examples include: Polyenes, Polymyxins, Nystatin

(iii) Inhibition of nucleic acid synthesis (either DNA or RNA synthesis). Examples
include: Fluoroquinolones, Rifamycins, Sulfonamides, Quinolones,
(iv) Inhibition of protein synthesis. Examples include: Aminoglycosides, Tetracyclines,
Chloramphenicol
(v) Inhibition of enzyme activity. Examples include: Penicillins, Cephalosporins,
Sulfonamides, Quinolones
(b) Based on the microbial group targeted

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 Antimicrobial agents may also be classified based on the general microbial group they act
against:
(i) Antibacterial. Examples include: Penicillin, Cephalosporins, Triclosan
(ii) Antifungal. Examples include: Polyenes, Fluconazole, Ketoconazole,
(iii) Antiprotozoan. Examples include: Chloroquine, Metronidazole(Flagyl), Mepron
(atovaquone), Pentamidine
(iv) Antiviral. Examples include: cyclovir, Oseltamivir, Zidovudine
(c) Based on the Spectrum of activity
(i) Narrow-spectrum drugs kill either Gram-positive or Gram-negative bacteria (they are
effective only against a limited variety of pathogens). Common examples of narrow-
spectrum antibiotics include Bacitracin, Streptomycin, Isoniazid
(ii) Broad-spectrum drugs kill both Gram-positives and Gram-negatives (attack many
different kinds of pathogens). Common examples of broad-spectrum antibiotics
include Amoxicillin, Ciprofloxacin, Azithromycin

(d) Based on mode of production

(i) Biosynthetic drugs: are produce naturally by living organisms. An example of a
biosynthetic antimicrobial drug is penicillin, which is produced by the fungus Penicillium
chrysogenum.

(ii) Semisynthetic drugs: they are natural antibiotics that have been structurally modified
by the addition of chemical groups to make them less susceptible to inactivation by
pathogens. An example of a semi-synthetic drug is amoxicillin, which is derived from
penicillin but has been modified to improve its stability and effectiveness

(iii) Synthetic drugs: are manufactured by chemical procedures independent of microbial

activity. Examples include: Sulfonamides, Meropenem, imipenem
(e) Based on their effect on microbial growth
Chemotherapeutic agents, like disinfectants, can be either cidal or static
(i) Static agents inhibit the growth of pathogens. Examples include: Tetracyclines,
Sulfonamides

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 (ii) Cidal agent kills the target pathogen. For example, penicillin is a bactericidal
antibiotic that kills bacteria by interfering with cell wall synthesis.

Antibiotics
An antibiotic is a substance produced by a microorganism that is effective in killing or inhibiting
the growth of other microorganisms. Antibiotics are produced by certain moulds and bacteria,
usually those that live in soil. The antibiotics produced by soil organisms give them a selective
advantage in the struggle for the available nutrients in the soil. Penicillin and cephalosporins are
examples of antibiotics produced by moulds; bacitracin, erythromycin, and chloramphenicol are
examples of antibiotics produced by bacteria. Although originally produced by microorganisms,
many antibiotics are now synthesized or manufactured in pharmaceutical laboratories.

Chemistry of Some Antibiotics of Medical Importance

(a) Penicillins
Since its accidental discovery in 1928, penicillin is the oldest known and still one of the most
widely used antibiotic agents. The penicillin group is characterized by the presence of the 4-
membered β-lactam ring fused to a 5-membered thiazolidine ring. In addition to the β-lactam and
thiazolidine rings, penicillins also have an acyl side chain attached to the β-lactam ring. The
nature of this side chain can vary among different penicillins and can affect their spectrum of
activity and resistance to bacterial enzymes that can degrade them, such as β-lactamases. The
precursor molecule to penicillins, known as 6-aminopenicillanic acid, contains only the β-lactam
and thiazolidine rings, with an amino group attached to the 6-carbon nucleus.
Penicillin antibiotics, based on the way they are synthesized, can be classified into two groups,
the naturally occurring and the semisynthetic penicillins.

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Figure 4. General Structure of Penicillin

(i) Naturally Occurring Penicillins

The naturally occurring penicillins are derived from certain species of the Penicillium fungi, with
Penicillium chrysogenum being one of the most common species used for the commercial
production of penicillins. Another species that was initially misidentified as the source of
penicillin is Penicillium notatum, which is now known as Penicillium rubens.
The two naturally occurring penicillins are Benzylpenicillin (penicillin G) and Phenoxymethyl
penicillin (penicillin V).

 Benzylpenicillin (penicillin G)

Figure 5. Structure of Benzylpenicillin (penicillin G)

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 Advantages
 Penicillin G has good tissue penetration
 High activity against most gram-positive bacteria
 Relatively non-toxic
 Relatively inexpensive in comparison to many other antibiotics
Limitations
 it is degraded by gastric acid and hence when used optimally it is usually given
parenterally (i.e., through injection)
 it is destroyed by the enzyme penicillinase (beta lactamase)
 Narrow spectrum of activity
Clinical Uses
Penicillin G is effective against Gram-positive cocci (e.g., Staphylococcus aureus, Streptococcus
pneumoniae, Streptococcus pyogenes), Gram-positive bacilli (e.g., Bacillus anthracis,
Corynebacterium diphtheriae), Gram-negative cocci (e.g., Neisseria gonorrhoeae, Neisseria
meningitidis), some anaerobic bacteria (e.g., Clostridium perfringens), and some spirochetes
(e.g., Treponema pallidum).

 Phenoxymethyl penicillin (penicillin V)

Figure 6. Structure of phenoxymethylpenicillin (penicillin V)

Advantages
 Penicillin V is relatively stable to gastric acid and can therefore be administered orally.
Limitations
 Penicillin V is a narrow-spectrum antibiotic
 It is destroyed by the enzyme beta lactamase

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 Clinical Uses
Penicillin-V is commonly prescribed for treatment of pharyngitis, skin infection, tonsillitis,
where infection with Streptococcus pyogenes is confirmed or suspected
Side effects of penicillin G and V
Penicillin V and G can have adverse effects, including nausea, vomiting, diarrhea, rash,
abdominal pain, and urticaria. In addition, Penicillin G can have other adverse reactions,
including muscle spasms, fever, chills, muscle pain, headache, tachycardia, flushing, tachypnea,
and hypotension.

(ii) Semisynthetic penicillins

In addition to naturally occurring penicillins such as penicillin G, many semisynthetic penicillins
have been developed. Semisynthetic penicillins are modified forms of natural penicillins that are
created by chemically altering the side chains (R groups) of the β-lactam ring. These
modifications can result in increased stability, broader spectrum of activity, and resistance to
enzymatic degradation.

Figure 7. Semisynthetic penicillins are created by chemically altering the side chains of the β-
lactam ring.

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The side chain of natural penicillin can be split off by an amidase to produce 6-aminopenicillanic
acid. The following structures can each be substituted at the R to produce new penicillin.

Figure 8. Semi-synthetic penicillins

(b) Cephalosporins
Cephalosporins are a family of antibiotics originally isolated in 1948 from the fungus
Cephalosporium (Cephalosporium acremonium). They contain a β-lactam structure that is very
similar to that of the penicillins. They are broad-spectrum drugs frequently given to patients with
penicillin allergies (although about 10% of patients allergic to penicillin are also allergic to
cephalosporins). Also like penicillins, cephalosporins interfere with cell wall synthesis and are
bactericidal.

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7-aminocephalosporanic acid is the starting material for the synthesis of most cephalosporins.
The chemical structure of 7-aminocephalosporanic acid contains a β-lactam ring fused to a
dihydrothiazine ring, which is the core structure of all cephalosporins. By modifying the side
chains attached to this core structure, a wide range of cephalosporins with different spectra of
activity and pharmacokinetic properties can be produced.

Figure 9. 7-aminocephalosporanic acid nucleus

Figure 10. These drugs are derivatives of 7-aminocephalosporanic acid and contain a β-
lactam ring.

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 Generations of Cephalosporin
Cephalosporins are classified into generations based on their spectrum of activity and
pharmacokinetic properties.
- The first-generation agents are Narrow-spectrum drugs, active primarily against Gram-
positive bacteria. Examples include: cefazolin, cephalothin, cephapirin, cephradine
- Second generation cephalosporins have increased activity against Gram-negative
bacteria. Examples include: cefamandole, cefonicid, cefuroxime
- Third-generation cephalosporins have even greater activity against Gram-negatives
(including Pseudomonas aeruginosa and members of the Enterobacteriaceae family).
Examples include: cefoperazone, cefotaxime, ceftazidime, ceftizoxime, ceftriaxone
- Fourth-generation cephalosporins are broad spectrum with excellent gram-positive and
gram-negative coverage and, like their third-generation predecessors, inhibit the growth
of the difficult opportunistic pathogen Pseudomonas aeruginosa e.g., cefepime.
- Fifth generation cephalosporins have a broad spectrum of activity against Gram-positive
and Gram-negative bacteria, including multi-drug-resistant strains such as methicillin-
resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. Examples
include: ceftaroline and ceftobiprole.
Adverse effects
Cephalosporins have low toxicity and are generally safe. The most common adverse reactions
from cephalosporins are nausea, vomiting, lack of appetite, and abdominal pain.

(c) Sulfonamides or Sulfa Drugs

The first antimetabolites to be used successfully as chemotherapeutic agents were the
sulfonamides. They are structural analogs of para-aminobenzoic acid (PABA), which is a
precursor to folic acid in bacteria. PABA is used in the synthesis of the cofactor folic acid
(folate). When sulfanilamide or another sulfonamide enters a bacterial cell, it competes with
PABA for the active site of an enzyme involved in folic acid synthesis, causing a decline in
folate concentration. This decline is detrimental to the bacterium because folic acid is a precursor
of purines and pyrimidines, the bases used in the construction of DNA, RNA, and other

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important cell constituents. The resulting inhibition of purine and pyrimidine synthesis leads to
cessation of protein synthesis and DNA replication, thus the pathogen dies

Figure 11. Structure similarity of sulfonamides and PABA

Figure 12: Two Sulfonamide Drugs

The Sulfonamides are bacteriostatic for some gram-negative and gram-positive bacteria,
chlamydiae, nocardia and protozoa.

Adverse effects
As many as 5% of the patients receiving sulfa drugs experience adverse side effects, chiefly
allergic responses such as fever, hives, and rashes.

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 (a) Quinolones
The quinolones are synthetic drugs that contain the 4-quinolone ring. The quinolones are
important antimicrobial agents that inhibit nucleic acid synthesis. They are increasingly used to
treat a wide variety of infections. The first quinolone, nalidixic acid was synthesized in 1962.
Since that time, generations of fluoroquinolones have been produced (e.g, ciprofloxacin,
norfloxacin, and ofloxacin). Quinolones are bactericidal drugs that inhibit DNA synthesis.
They are broad-spectrum antibiotics.

Figure 13. quinolones

Clinical Uses
Quinolones are highly effective against enteric bacteria such as E. coli and Klebsiella
pneumoniae. They can be used with Haemophilus, Neiserria, P. aeruginosa, and other gram-
negative pathogens. The quinolones also are active against gram-positive bacteria such as S.
aureus, Streptococcus pyogenes, and Mycobacterium tuberculosis. They are used in treating
urinary tract infections, sexually transmitted diseases caused by Neisseria and Chlamydia,
gastrointestinal infections, respiratory infections, skin infections, and osteomyelitis (bone
infection).
Adverse effect
The most prominent adverse effects are nausea, insomnia, headache, and dizziness.

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 Assignment
Write an in-depth essay about the production of penicillin, covering both
upstream and downstream processing stages

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 Antiviral agents
Antiviral agents are specifically designed to target viral infections. Antiviral agents are
particularly difficult to develop and use because viruses are produced within host cells. They
target specific viral components or processes to prevent viral replication and control viral
infections. Different classes of antiviral agents target specific viruses and mechanisms of action,
such as blocking viral fusion, inhibiting viral enzymes, or preventing viral gene transcription
(a) Entry inhibitors/fusion inhibitors
These drugs target the initial stages of viral infection by preventing the entry of the virus into
host cells. They can block viral attachment or fusion with host cell membranes. Entry inhibitors
are used in the treatment of human immunodeficiency virus (HIV), such as maraviroc, which
works by binding to the CCR5 chemokine receptor on the surface of CD4+ T cells, thereby
preventing the entry of HIV-1 into these cells. Also another fusion inhibitor called Enfuvirtide
(T-20) targets the gp41 subunit of the HIV envelope protein, preventing the virus from entering
CD4+ T cells.
(b) Protease Inhibitors
Protease inhibitors are a class of antiviral drugs that work by blocking the activity of viral
proteases, enzymes that are essential for maturation of viral proteins and viral replication. By
blocking protease activity, these drugs prevent the formation of functional viral proteins, thus
inhibiting viral replication. Protease inhibitors are commonly used in the treatment of HIV, such
as ritonavir and darunavir. Protease inhibitors are also used in the treatment of chronic hepatitis
C virus (HCV) infection
(c) Nucleoside reverse Transcriptase Inhibitors
Reverse transcriptase is an enzyme used by retroviruses, like HIV, to convert their RNA
genome into DNA. Reverse transcriptase inhibitors block this enzymatic activity, preventing the
conversion of viral RNA into DNA and thus inhibiting viral replication. Nucleoside reverse
transcriptase inhibitors are nucleoside analogues that are incorporated into the growing viral
DNA chain during the reverse transcription process. Once incorporated, they compete with
natural nucleotides for incorporation into the DNA chain, thereby causing chain termination and
preventing the formation of a complete viral DNA molecule. Examples include: zidovudine
(AZT) and lamivudine.

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 (d) Neuraminidase Inhibitors
Neuraminidase is an enzyme produced by influenza viruses that allows the virus to be released
from infected host cells and spread to other cells. Without active neuraminidase, the virus cannot
cleave sialic acid and escape from the cell. Neuraminidase inhibitors, such as oseltamivir and
zanamivir, block the activity of this enzyme, reducing the release and spread of influenza
viruses.

Figure 14. Antiviral agents

Antifungal agents

An antifungal agent is a medication or drug that is specifically designed to target and eliminate
fungal pathogens from a host while minimizing toxicity to the host's cells. Fungal infections, also
known as mycoses, can affect various parts of the body, including the skin, nails, mucous
membranes, and internal organs. Antifungal agents work by targeting specific components or
processes unique to fungi, thereby disrupting their growth, metabolism, or reproduction.

Most antifungal agents work in one of these ways:

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 (a) By binding with cell membrane sterols (e.g., nystatin and amphotericin B).

In fungi, the dominant sterol is ergosterol; in mammalian cells, it is cholesterol. The polyenes
nystatin and amphotericin B are lipophilic and bind to sterols in the cytoplasmic membrane of
eukaryotic cells. Following binding, they form annular channels, which penetrate the membrane
and lead to leakage of essential small molecules from the cytoplasm and cell death. The basis of
their selective toxicity is their greater affinity for the sterols of fungal membranes, such as
ergosterol, than the sterols of human cells

(b) By interfering with sterol synthesis

The azoles are a large family of synthetic organic compounds, which includes members with
antibacterial, antifungal, and antiparasitic properties. The important antifungal azoles are the
imidazole, ketoconazole, and the triazoles, fluconazole and itraconazole. Others are under
development or evaluation. Their activity is based on inhibition of a cytochrome enzyme (P450
demethylase) responsible for conversion of lanosterol to ergosterol, the major component of the
fungal cytoplasmic membrane. This leads to lanosterol accumulation and the formation of a
defective cell membrane with altered permeability characteristics.

(c) By interfering with cell wall synthesis

The fungal cell wall is composed of layers including mannan, mannoprotein, glucan, and chitin,
making it a unique target for antifungal treatments. Echinocandins are a class of agents that
inhibit glucan synthesis, causing effects in yeast similar to those caused by β-lactams in bacteria.
Caspofungin, the first licensed echinocandin, is effective against Candida and Aspergillus.
Another class, the nikomycins, targets chitin synthesis but is still in early development.

(d) By blocking nucleic acid synthesis or mitosis

5-flucytosine is converted to 5-fluorouracil within fungal cells. This interferes with DNA and
RNA synthesis, ultimately inhibiting fungal cell division and growth. Griseofulvin works by
inhibiting the growth of fungi. It does this by disrupting the formation of the fungal cell wall,
which is essential for the fungus's survival and replication.

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 Antiprotozoal agents
Antiprotozoal agents are medications or drugs that are specifically designed to target and
eliminate protozoal infections.
Antiprotozoal drugs are usually quite toxic to the host and work by:
(a) Interfering with DNA and RNA synthesis (e.g., chloroquine, pentamidine, and quinacrine).
(b) Interfering with protozoal metabolism (e.g., metronidazole; brand name Flagyl).

Figure 16. Examples of antiprotozoal agents

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