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Important Protozoan Pathogens
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• Infectious ciliates
– Balantidium coli–a
zoonotic disease
acquired by humans
via the fecal-oral
route from the
normal host, the pig,
where it is
asymptomatic.
Contaminated water
is the most common
mechanism of
transmission.
.
Apicomplexa
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Cytostome
Food vacuoles
Nucleus
Cell membrane
Cytostome
(mouth)
Food
vacuole
Nucleus
Endoplasmic
reticulum
Mitochondrion
©
(a)
ASM
Michael Riggs et al, Infection and Immunity, Vol. 62, #5, May 1994, p. 1931
(b)
2 2
Important Protozoan Pathogens
• Infectious
apicomplexansPlasmodium species:
Cause of malaria;
• Toxoplasma gondii:
Cause of
Toxoplasmosis
• Cryptosporidium sp.:
Cause of
Cryptosporidiosis
Parasitic Helminths
• Multicellular animals; organs for
reproduction, digestion, movement, protection
• Parasitize host tissues
• Have mouthparts for attachment to or
digestion of host tissues
• Most have well-developed sex organs that
produce eggs and sperm
• Fertilized eggs go through larval period in or
out of host body
4
Helminth Classification and Identification
• Classify according to shape, size, organ development,
presence of hooks, suckers, or other special structures, mode
of reproduction, hosts, and appearance of eggs and larvae
• Identify by microscopic detection of worm, larvae, or eggs
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Oral sucker
Pharynx
Esophagus
Intestine
Ventral
sucker
Cuticle
Vas deferens
Uterus
Cuticle
Ovary
Testes
Scolex
(a)
Seminal
receptacle
Proglottid
Suckers
Immature eggs
Fertile eggs
(b)
Excretory
bladder
5
Major Groups of Parasitic Helminths
1. Flatworms – flat, no definite body cavity;
digestive tract a blind pouch; simple excretory
and nervous systems
•
•
Cestodes (tapeworms)
Trematodes or flukes, are flattened, nonsegmented
worms with sucking mouthparts
2. Roundworms (nematodes) – round, a
complete digestive tract, a protective surface
cuticle, spines and hooks on mouth; excretory
and nervous systems poorly developed
6
The Position of Viruses in the Biological
Spectrum
• There is no universal
agreement on how and
when viruses originated
• Viruses are considered
the most abundant
microbes on earth
• Viruses played a role in
the evolution of Bacteria,
Archaea, and Eukarya
• Viruses are obligate
intracellular parasites
7
General Structure of Viruses
• Capsids
– All viruses have capsids
(protein coats that
enclose and protect their
nucleic acid)
– The capsid together with
the nucleic acid is the
nucleocapsid
– Some viruses have an
external covering called
an envelope; those
lacking an envelope are
naked
– Each capsid is made of
identical protein subunits
called capsomers
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Capsid
Nucleic acid
(a) Naked Nucleocapsid Virus
Envelope
Spike
Capsid
Nucleic acid
8
(b) Enveloped Virus
Types of Viruses
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A. Complex Viruses
B. Enveloped Viruses
Helical
Icosahedral
(1)
(3)
(5)
(2)
(4)
(6)
C. Nonenveloped Naked Viruses
Helical
Icosahedral
(8)
(7)
(9)
A. Complex viruses:
(1) poxvirus, a large DNA virus
(2) flexible-tailed bacteriophage
B. Enveloped viruses:
With a helical nucleocapsid:
(3) mumps virus
(4) rhabdovirus
With an icosahedral nucleocapsid:
(5) herpesvirus
(6) HIV (AIDS)
C. Naked viruses:
Helical capsid:
(7) plum poxvirus
Icosahedral capsid:
(8) poliovirus
(9) papillomavirus
9
Nucleic Acids
• DNA viruses
– Usually double stranded (ds) but may be single
stranded (ss)
– Circular or linear
• RNA viruses
– Usually single stranded, may be double stranded, may
be segmented into separate RNA pieces
– ssRNA genomes ready for immediate translation are
positive-sense RNA
– ssRNA genomes that must be converted into proper
form are negative-sense RNA
10
Modes of Viral Multiplication
General phases in animal virus multiplication cycle:
1. Adsorption – binding of virus to specific molecules on
the host cell
2. Penetration – genome enters the host cell
3. Uncoating – the viral nucleic acid is released from the
capsid
4. Synthesis – viral components are produced
5. Assembly – new viral particles are constructed
6. Release – assembled viruses are released by
budding (exocytosis) or cell lysis
11
Adsorption and Host Range
• Virus coincidentally collides with a susceptible host cell and
adsorbs specifically to receptor sites on the membrane
• Spectrum of cells a virus can infect – host range
– Hepatitis B – human liver cells
– Poliovirus – primate intestinal and nerve cells
– Rabies – various cells of many mammals
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Envelope spike
Host cell membrane
Capsid spike
Receptor
Host cell
membrane
Receptor
12
(a)
(b)
Replication and Protein Production
• Varies depending on whether the virus is a
DNA or RNA virus
• DNA viruses generally are replicated and
assembled in the nucleus
• RNA viruses generally are replicated and
assembled in the cytoplasm
– Positive-sense RNA contain the message for
translation
– Negative-sense RNA must be converted into
positive-sense message
13
Release
• Assembled viruses leave the host
cell in one of two ways:
– Budding – exocytosis;
nucleocapsid binds to membrane
which pinches off and sheds the
viruses gradually; cell is not
immediately destroyed
– Lysis – nonenveloped and
complex viruses released when
cell dies and ruptures
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(b)
© Chris Bjornberg/Photo Researchers, Inc.
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Host cell membrane
Viral nucleocapsid
Viral glycoprotein spikes
Cytoplasm
Capsid
RNA
Budding
virion
(a)
Viral matrix
protein
Free infectious
virion with envelope
14
Damage to Host Cell
Cytopathic effects (CPE) –
virus-induced damage to cells
1. Changes in size and shape
2. Cytoplasmic inclusion
bodies
3. Inclusion bodies
4. Cells fuse to form
multinucleated cells
5. Cell lysis
6. Alter DNA
7. Transform cells into
cancerous cells
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Multiple
nuclei
Normal
cell
Giant cell
CDC
(a)
CDC
Inclusion bodies
15
(b)
© Massimo Battaglia, INeMM CNR, Rome Italy
Viral Damage
• Some animal viruses enter the host cell and
permanently alter its genetic material resulting in
cancer – transformation of the cell
• Transformed cells have an increased rate of
growth, alterations in chromosomes, and the
capacity to divide for indefinite time periods
resulting in tumors
• Mammalian viruses capable of initiating tumors
are called oncoviruses
– Papillomavirus – cervical cancer
– Epstein-Barr virus – Burkitt’s lymphoma
16
Lysogeny: The Silent Virus Infection
• Not all phages complete the lytic cycle
• Some DNA phages, called temperate phages,
undergo adsorption and penetration but don’t replicate
• The viral genome inserts into bacterial genome and
becomes an inactive prophage – the cell is not lysed
• Prophage is retained and copied during normal cell
division resulting in the transfer of temperate phage
genome to all host cell progeny – lysogeny
• Induction can occur resulting in activation of lysogenic
prophage followed by viral replication and cell lysis
17
Prions Diseases
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Common in animals:
• Scrapie in sheep
and goats
• Bovine
spongiform
encephalopathies
(BSE), a.k.a. mad
cow disease
• Wasting disease in
elk
• Humans –
Creutzfeldt-Jakob
Syndrome (CJS)
Brain cell
Prion fibrils
© James King-Holmes/Institute of Animal Health/Photo Researchers, Inc.
(a)
18
Dr. Art Davis/CDC
(b)
Other Noncellular Infectious Agents
•
Satellite viruses – dependent on other viruses
for replication
– Adeno-associated virus – replicates only in cells
infected with adenovirus
– Delta agent – naked strand of RNA expressed only
in the presence of hepatitis B virus
•
Viroids – short pieces of RNA, no protein coat;
only been identified in plants
19
The Metabolism of Microbes
Two types of chemical reactions:
Catabolism – degradative;
breaks the bonds of larger
molecules forming smaller
molecules; releases energy
Anabolism – biosynthesis;
process that forms larger
macromolecules from
smaller molecules; requires
energy input
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Synthesis of Cell Structures
Macromolecules
Carbohydrates
Proteins
Lipids
Sources of energy
Carbohydrates
Lipids
Proteins
Catabolism
Releases energy
End products with
reduced energy
CO2, H2O
ATP
NADH
Anabolism
Requires energy
Simple building
blocks
Sugars
Amino acids
Increasing complexity
Metabolism – all chemical and
physical workings of a cell
20
Enzymes
• Enzymes are biological catalysts that increase the rate of a
chemical reaction by lowering the energy of activation (the
resistance to a reaction)
• The enzyme is not permanently altered in the reaction
• Enzyme promotes a reaction by serving as a physical site
for specific substrate molecules to position
Energy State of Reaction
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Reactant
Energy of activation in the
absence of enzyme
Energy of activation in the
presence of enzyme
Initial
state
Products
Final state
Progress of Reaction
21
Enzyme Structure
• Simple enzymes – consist of protein alone
• Conjugated enzymes or holoenzymes –
contain protein and nonprotein molecules
– Apoenzyme – protein portion
– Cofactors – nonprotein portion
• Metallic cofactors: iron, copper, magnesium
• Coenzymes, organic molecules: vitamins
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Coenzyme
Coenzyme
Metallic
cofactor
Apoenzymes
Metallic
cofactor
22
Cofactors: Supporting the Work of Enzymes
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Chemical
group (Ch)
Substrate 2 (S2 )
• Micronutrients
are needed as
cofactors
• Cofactors act
as carriers to
assist the
enzyme in its
activity
1. An enzyme
with a coenzyme
positioned to
react with two
substrates.
Substrate 1 (S1)
Coenzyme
(C)
Enzyme
complex
(E)
Ch
S2
2. Coenzyme
picks up a
chemical group
from substrate 1.
C
E
S2
3. Coenzyme
readies the
chemical group
for transfer to
substrate 2.
S1
Ch
S1
C
E
Ch S1
4. Final action
is for group to
be bound to
substrate 2;
altered substrates
are released
from enzyme.
C
E
S1
23
Regulation of enzyme action
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Noncompetitive Inhibition
Competitive Inhibition
Normal
substrate
Substrate
Competitive
inhibitor with
similar shape
Active site
Both molecules
compete for
the active site.
Regulatory site
Enzyme
Regulatory
molecule
(product)
Enzyme
Reaction proceeds
Enzyme
Reaction is blocked
because competitive
inhibitor is incapable
of becoming a product.
Reaction proceeds
Product
Reaction is blocked because
binding of regulatory molecule in
regulatory site changes
conformation of active site so
that substrate cannot enter .
24
Enzyme repression
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1
DNA
2
3
RNA
4 Folds to form functional
enzyme structure
Protein
Enzyme
+
Substrate
5
Products
6
7
DNA
RNA
Excess product binds to
DNA and shuts down further
enzyme production.
Protein
No enzyme
25
Biological Oxidation and Reduction
• Redox reactions – always occur in pairs
• There is an electron donor and electron
acceptor which constitute a redox pair
• Process salvages electrons and their energy
• Released energy can be captured to
phosphorylate ADP or another compound
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Oxidation
C6H12O6 + 6O2 → 6CO2 + H2O + Energy
h
Glucose
Reduction
26
Pathways of Bioenergetics
•
•
•
Bioenergetics – study of the mechanisms of
cellular energy release
Includes catabolic and anabolic reactions
Primary catabolism of fuels (glucose) proceeds
through a series of three coupled pathways:
1. Glycolysis
2. Kreb’s cycle
3. Respiratory chain, electron transport
27
Metabolic Strategies
• Nutrient processing is varied, yet in many cases is
based on three catabolic pathways that convert glucose
to CO2 and gives off energy
• Aerobic respiration – glycolysis, the Kreb’s cycle,
respiratory chain
• Anaerobic respiration – glycolysis, the Kreb’s cycle,
respiratory chain; molecular oxygen is not the final
electron acceptor
• Fermentation – glycolysis, organic compounds are the
final electron acceptors
28
Biosynthesis and the Crossing
Pathways of Metabolism
• Many pathways of metabolism are bi-directional or amphibolic
Anabolism
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Chromosomes
Enzymes
Membranes
Cell wall
Storage
Membranes
Storage
Nucleic
acids
Proteins
Starch
Cellulose
Lipids
Fats
Nucleotides
Amino acids
Carbohydrates
Fatty acids
Cell
structure
Macromolecule
Building block
Beta
oxidation
Deamination
Glycolysis
GLUCOSE
Metabolic
pathways
Catabolism
Pyruvic acid
Acetyl coenzyme A
Krebs
cycle
NH3
H2O
CO2
Simple
products
29
Overview of Catabolic Pathways
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(a)
AEROBIC RESPIRATION
(b) ANAEROBIC RESPIRATION
Glycolysis
Glucose
(c)
FERMENTATION
Glycolysis
(6 C)
ATP
Glucose
Glycolysis
(6 C)
ATP
NADH
NADH
2 pyruvate (3 C)
CO2
CO2
Acetyl Co A
2 pyruvate (3 C)
CO2
Fermentation
Acetyl Co A
FADH2
(6 C)
ATP
NADH
2 pyruvate (3 C)
Glucose
FADH2
Krebs
NADH
CO2
Krebs
CO2
Lactic acid
Acetaldehyde
NADH
ATP
Electrons
Electron transport
O2is final electron
acceptor.
Maximum
ATP produced = 38
ATP
Electrons
Ethanol
+CO2
Or other alcohols,
acids, gases
Electron transport
Nonoxygen electron acceptors
(examples: SO42–, NO3–,CO32–)
Maximum
ATP produced = 2 – 36
An organic molecule is final
electron acceptor (pyruvate,
acetaldehyde, etc.).
Maximum
ATP produced = 2
30
Electron Transport and Oxidative
Phosphorylation
• Final processing of electrons and hydrogen and
the major generator of ATP
• Chain of redox carriers that receive electrons
from reduced carriers (NADH and FADH2)
• ETS shuttles electrons down the chain, energy is
released and subsequently captured and used
by ATP synthase complexes to produce ATP –
Oxidative phosphorylation
31
The Formation of ATP and
Chemiosmosis
• Chemiosmosis – as the electron transport carriers
shuttle electrons, they actively pump hydrogen ions
(protons) across the membrane setting up a gradient of
hydrogen ions – proton motive force
• Hydrogen ions diffuse back through the ATP synthase
complex causing it to rotate, causing a 3-D change
resulting in the production of ATP
32
Photosynthesis
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• Occurs in 2 stages:
• Light-dependent –
photons are absorbed
by chlorophyll,
carotenoid, and
phycobilin pigments
– Water split by
photolysis, releasing
O2 gas and provides
electrons to drive
photophosphorylation
– Released light energy
used to synthesize
ATP and NADPH
Glucose
H2O
ATP
2H + e2
NADPH
O2
Chloroplast
CO2
33
Photosynthesis Reactions
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
(a) A cell of the motile alga Chlamydomonas, with a single
large chloroplast (magnified cutaway view). The chloroplast
contains membranous compartments called grana where
chlorophyll molecules and the photosystems for the light
reactions are located.
Flagellum
Nucleus
Chloroplast
Cell wall
A
Stroma
B
N
Granum
N
Mg
N
D
N
C
(b) A chlorophyll molecule,
with a central magnesium
atom held by a porphyrin ring.
Photons
(c) The main events of the light reactions shown as an exploded view in
one granum.
2
2
e–
NADP
5
P700
1
photolysis
1/2 O2
2 e–
H+
3
H+
H+
H 2O
NADPH
Photosystem I
P680
2H+
Photosystem II
Thylakoid membrana
H+
ADP + Pi
6
ATP
H+
ATP
synthase
Proton
pump
1
When light activates photosystem II, it sets up a chain reaction, in
which electrons are released from chlorophyll.
2
These electrons are transported along a chain of carriers to
photosystem I.
3
The empty position in photosystem II is replenished by photolysis of
H2O. Other products of photolysis are O2 and H+.
4
Pumping of H into the interior of the granum produces conditions for
ATP to be synthesized.
5
The final electron and H acceptor is NADP, which receives these from
photosystem I.
6
Both NADPH and ATP are fed into the stroma for the Calvin cycle.
H+
Calvin Cycle
2 e–
Interor of granum
4
H+
34
Controlling Microorganisms
• Physical, chemical, and mechanical methods
to destroy or reduce undesirable microbes in
a given area (decontamination)
• Primary targets are microorganisms capable
of causing infection or spoilage:
–
–
–
–
–
–
Vegetative bacterial cells and endospores
Fungal hyphae and spores, yeast
Protozoan trophozoites and cysts
Worms
Viruses
Prions
35
Relative Resistance of Microbes
• Highest resistance
– Prions, bacterial endospores
• Moderate resistance
–
–
–
–
Pseudomonas sp.
Mycobacterium tuberculosis
Staphylococcus aureus
Protozoan cysts
• Least resistance
–
–
–
–
Most bacterial vegetative cells
Fungal spores and hyphae, yeast
Enveloped viruses
Protozoan trophozoites
36
Terminology and Methods of Control
• Sterilization – a process that destroys all viable
microbes, including viruses and endospores
• Disinfection – a process to destroy vegetative
pathogens, not endospores; inanimate objects
• Antiseptic – disinfectants applied directly to
exposed body surfaces
• Sanitization – any cleansing technique that
mechanically removes microbes
• Degermation – reduces the number of microbes
through mechanical means
37
Practical Concerns in Microbial Control
Selection of method of control depends on
circumstances:
• Does the application require sterilization?
• Is the item to be reused?
• Can the item withstand heat, pressure, radiation,
or chemicals?
• Is the method suitable?
• Will the agent penetrate to the necessary extent?
• Is the method cost- and labor-efficient and is it
safe?
38
Antimicrobial Agents’ Modes of Action
Cellular targets of physical and chemical agents:
1. The cell wall – cell wall becomes fragile and
cell lyses; some antimicrobial drugs,
detergents, and alcohol
2. The cell membrane – loses integrity; detergent
surfactants
39
Methods of Physical Control
1.
2.
3.
4.
5.
Heat – moist and dry
Cold temperatures
Desiccation
Radiation
Filtration
40
Chemical Agents in
Microbial Control
• Disinfectants, antiseptics, sterilants,
degermers, and preservatives
• Some desirable qualities of chemicals:
–
–
–
–
–
–
Rapid action in low concentration
Solubility in water or alcohol, stable
Broad spectrum, low toxicity
Penetrating
Noncorrosive and nonstaining
Affordable and readily available
41
Germicidal Categories
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Halogens
Phenolics
Chlorhexidine
Alcohols
Hydrogen peroxide
Aldehydes
Gases
Detergents & soaps
Heavy metals
Dyes
Acids and Alkalis
42
Principles of Antimicrobial
Therapy
• Administer a drug to an infected person that
destroys the infective agent without harming the
host’s cells
• Antimicrobial drugs are produced naturally or
synthetically
43
Mechanisms of Drug Action
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4. Protein synthesis inhibitors acting
1. Cell wall inhibitors
Block synthesis and repair
Penicillins
Cephalosporins
Vancomycin
Bacitracin
Monobactams/carbapenems
Fosfomycin
Cycloserine
Isoniazid
on ribosomes
Ribosome
Site of action
50S subunit
Chloramphenicol
Erythromycin
Clindamycin
Streptogramin (Synercid)
Substrate
2. Cell membrane
Enzyme
Cause loss of selective permeability
Polymyxins
Product
3. DNA/RNA
Inhibit replication and transcription
Inhibit gyrase (unwinding enzyme)
Quinolones (ciprofloxacin)
Inhibit RNA polymerase
Rifampin
Site of action
30S subunit
Aminoglycosides
Gentamicin
Streptomycin
Tetracyclines
Both 30S
and 50S
mRNA
DNA
Blocks initiation of protein
Synthesis
Linezolid (Zyvox)
5. Metabolic pathways and products
Block pathways and inhibit
Metabolism
Sulfonamides (sulfa drugs)
Trimethoprim
44
Antibacterial Drugs that
Act on the Cell Wall
• Beta-lactam antimicrobials - all contain a highly
reactive 3 carbon, 1 nitrogen ring
• Primary mode of action is to interfere with cell wall
synthesis
• Greater than ½ of all antimicrobic drugs are betalactams
• Penicillins and cephalosporins most prominent betalactams
ß-Lactams: Mechanisms of Action and Resistance
https://www.youtube.com/watch?v=qBdYnRhdWcQ
45
Antimicrobial Drugs That Affect the
Bacterial Cell Wall
• Most bacterial cell walls contain peptidoglycan
• Penicillins and cephalosporins block synthesis of
peptidoglycan, causing the cell wall to lyse
• Active on young, growing cells
• Penicillins that do not penetrate the outer membrane
and are less effective against gram-negative bacteria
• Broad spectrum penicillins and cephalosporins can
cross the cell walls of gram-negative bacteria
46
Penicillin and Its Relatives
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• Large diverse group of
compounds
• Could be synthesized in
the laboratory
• More economical to obtain
natural penicillin through
microbial fermentation and
modify it to semi-synthetic
forms
• All consist of 3 parts:
– Thiazolidine ring
– Beta-lactam ring
– Variable side chain
dictating microbial
activity
Nucleus
(Aminopenicillanic acid)
R Group
Betalactam
Thiazolidine
S
CO
N
CH3
CH3
N
O
Nafcillin
COOH
S
CH
N
CO
CH3
CH3
COONa
N
O
COOH
S
Ticarcillin
Cl
S
CO
N
CH3
CH3
N
O
N
O
COOH
CH3
Cloxacillin
S
CH
CO
N
CH3
CH3
COONa
Carbenicillin
N
O
COOH
Cephalosporins
• 4 generations exist: each group more effective
against gram-negatives than the one before with
improved dosing schedule and fewer side effects
– First generation – cephalothin, cefazolin – most
effective against gram-positive cocci and few gramnegative
– Second generation – cefaclor, cefonacid – more
effective against gram-negative bacteria
– Third generation – cephalexin, ceftriaxone – broadspectrum activity against enteric bacteria with betalactamases
– Fourth generation – cefepime – widest range; both
gram- negative and gram-positive
48
Non Beta-lactam Cell Wall
Inhibitors
• Vancomycin – narrow-spectrum, most effective in
treatment of Staphylococcal infections in cases of
penicillin and methicillin resistance or if patient is allergic
to penicillin; toxic and hard to administer; restricted use
• Bacitracin – narrow-spectrum produced by a strain of
Bacillus subtilis; used topically in ointment
• Isoniazid (INH) – works by interfering with mycolic acid
synthesis; used to treat infections with Mycobacterium
tuberculosis
49
Antimicrobial Drugs That Disrupt Cell
Membrane Function
• A cell with a damaged membrane dies from disruption
in metabolism or lysis
• These drugs have specificity for a particular microbial
group, based on differences in types of lipids in their
cell membranes
• Polymyxins interact with phospholipids and cause
leakage, particularly in gram-negative bacteria
• Amphotericin B and nystatin form complexes with
sterols on fungal membranes which causes leakage
50
51