Monday, November 2, 2009
John Martinko, Department of Microbiology
Room 126 LS II
536-2349
Office Hours: M, W 2-5, or by appointment
Web page:
http://www.science.siu.edu/microbiology/micr453/
Primary resource:
Brock Biology of Microorganisms, 12th edition
Madigan, Martinko, Dunlap, and Clark, 2009
Pearson-Benjamin Cummings, San Francisco, CA
ISBN
0-132-32460-1
Brock Biology of Microorganisms
12 th
Edition
Chapter 27:
Microbial Growth Control
Sections 27.1-3
I. Physical antimicrobial control
Heat – autoclave ( F 27.3), Pasteurization, flame (loop)
Radiation - gamma rays, microwave, UV
Filtration –
II. Chemical Antimicrobial Control
27.4 Chemical Growth Control
Chemical agents are routinely used to control microbial growth.
antimicrobial agent: a natural or synthetic chemical that kills
or inhibits the growth of microorganisms.
-cidal agents kill microorganisms
prefix indicates the kind of organism killed
bacteriocidal, fungicidal, and viricidal agents
-static agents inhibit microbial growth
bacteriostatic, fungistatic, and viristatic agents
Some -cidal agents may also be
-lytic agents - lyse micoorganisms and viruses
Effect of Chemical Antimicrobial Agents on Growth
Chemicals that have toxicity limit microbial growth in vitro.
Experiment :
Chemical antimicrobial agents are added to exponentially
growing bacterial cultures: Results: Figure 27.9
bacteriostatic, inhibitors of protein synthesis, act by binding to
ribosomes - tetracycline
bacteriocidal, agents bind tightly to cellular targets and are not
removed by dilution - penicillin
bacteriolytic agents induce killing by cell lysis, - a decrease in cell
number and turbidity - detergents dissolve membranes
penicillin
Measuring Antimicrobial Activity (Figure 27.10)
minimum inhibitory concentration (MIC) - the lowest amount of agent
needed to completely inhibit the growth of a test organism (tube dilution
technique)
Figure 27.11
Agar medium is inoculated with the test organism.
Known amounts of antimicrobial agent are added
to filter paper disks and placed on the surface of the agar.
The agent diffuses from the filter paper into the agar; the further from the filter paper, the lower the concentration of the agent.
The diameter of the zone of inhibition is proportional to the amount of antimicrobial agent
added to the disk, the solubility, the diffusion coefficient, and the overall antimicrobial effectiveness.
¥Distinguish between -static, -cidal, and -lytic agents.
¥Describe how the minimum inhibitory concentration of an antibacterial agent is determined
27.5 Chemical
Antimicrobial Agents for External Use
1. We must control microorganisms not pathogenic in humans
Table 27.3 - mostly industrial applications
2.We must control growth of human pathogens in vitro
(on inanimate objects).
sterilants, disinfectants, sanitizers, and antiseptics.
Table 27.4 – healthcare, food, water, personal hygiene
Table 27.4 Sterilants are used on inanimate objects
(not live tissue)
sterilants, / sterilizers / sporicides destroy all forms of
(microbial) life, -even endospores AND viruses
Chemical sterilants are used when it is impractical to use heat or
radiation
Hospital, laboratory applications:
thermometers, lensed instruments, tubing, catheters,
reusable medical equipment (respirometers)
cold sterilization - enclosed devices that resemble autoclaves
employ a gaseous chemical agent:
ethylene oxide, formaldehyde, peroxyacetic acid, or
hydrogen peroxide
Liquid sterilization -sodium (hypo)chlorite solution / amylphenol
instruments that cannot withstand high temperatures or gas
Table 27.4
Disinfectants, Sanitizers
kill microorganisms, but not all kill endospores,
used on inanimate objects
Disinfectants- kill (almost) all microorganisms - proper mix and
exposure
Hospital infection control
-ethanol and cationic detergents
decontaminate floors, tables, bench tops, walls
Household disinfectants for general disinfection
swimming pools, and water purifiers
Sanitizers - reduce, but may not eliminate, microbial numbers to
a "safe" level- water soluble
Food contact sanitizers - mixing and cooking equipment,
dishes, and utensils
non-food contact sanitizers - counters, floors, walls, carpets,
air, and laundry
Antiseptics and germicides- chemical agents that kill or inhibit
growth of microorganisms and are sufficiently nontoxic to be
applied to living tissues.
handwashing (hand ÒsanitizersÓ/ treating surface wounds
certain antiseptics may also be effective disinfectants (FDA)
Antimicrobial Efficacy
Disinfectants and others can be neutralized by organic materials
e.g., pathogens are often grow in large numbers as
biofilms, limiting penetration of a chemical agent
ONLY sterilants are effective against bacterial endospores
Certain vegetative cells - Mycobacterium tuberculosis-
are resistant to the action of common disinfectants
because of the nature of their cell wall
¥Distinguish between sterilizer, a disinfectant, a sanitizer, and an antiseptic.
¥What disinfectants are routinely used for treatment of water? Why are these disinfectants not harmful to humans?
Physical / chemical growth control
Sterilizers
Kill
all (even spores) – 37% formaldehyde
Disinfectants
Kill
all but spores – surfaces
3-8%
formaldehyde, Lysol, high bleach
Sanitizer
Kill
most – reduce microbial load - not spores - surfaces
- low bleach
Antiseptics (germicides)
Kill
most
Safe
for contact with skin –
alcohols
reduce
microbial load
Selective toxicity
Growth factors / Analogs
III. Antimicrobial Agents Used in vivo
Treatment and Prevention of Infectious Disease
Compounds for internal use
Synthetic drugs
Antibiotics
27.6 Synthetic Antimicrobial Drugs
-manufactured chemical compounds that can be used internally
selective toxicity - the ability to inhibit or kill pathogenic
microorganisms without adversely affecting the host
First example - proof of concept Fig 27.15
Bacterial structures are different from eukaryotic structures
-targets for antimicrobial therapy Fig 27.12
Some microorganisms are more susceptible to certain agents
than others - different structures, etc.
Fig 27.13
27.6 Synthetic Antimicrobial Drugs
-manufactured chemical compounds that can be used internally
selective toxicity - the ability to inhibit or kill pathogenic
microorganisms without adversely affecting the host
First example - proof of concept Fig 27.15
Bacterial structures are different from eukaryotic structures
-targets for antimicrobial therapy Fig 27.12
Some microorganisms are more susceptible to certain agents
than others - different structures, etc.
Fig 27.13
27.6 Synthetic Antimicrobial Drugs
-manufactured chemical compounds that can be used internally
selective toxicity - the ability to inhibit or kill pathogenic
microorganisms without adversely affecting the host
First example - proof of concept Fig 27.15
Bacterial structures are different from eukaryotic structures
-targets for antimicrobial therapy Fig 27.12
Some microorganisms are more susceptible to certain agents
than others - different structures, etc.
Fig
27.13
Broad spectrum
Narrow spectrum
27.6 Synthetic Antimicrobial Drugs
Growth factor analogs
Nucleic acid base analogs
Quinolones
Growth factor analogs
-synthetic compounds, structurally similar to a growth factor
-subtle structural differences between the analogs and the
authentic growth factors prevent the analogs from functioning
in the cell
GF analogs for vitamins, amino acids, purines,
pyrimidines, and other compounds
Sulfa Drugs
-first widely used growth factor analogs that specifically
inhibit the growth of bacteria
-predate antibiotics
-large-scale screening of chemicals to find a cure
for streptococcal diseases in experimental animals
Figure 27.16 Sulfanilamide
p-aminobenzoic acid (PABA) analog - part of the folic acid,
vitamin - nucleic acids and amino acids
Blocks folic acid synthesis, inhibiting nucleic acid synthesis
Active only in Bacteria
Bacteria must synthesize folic acid
most animals obtain folic acid from their diet
Widespread drug resistance developed with time - 10-20 years
-sulfamethoxazole (a clinically useful sulfa drug) plus trimethoprim,
a related folic acid synthesis competitor, is now used
-drug combination produces sequential blocking of the
folic acid synthesis pathway
-resistance to the combination drug requires two mutations
in the same pathway in the same organism
Nucleic Acid base analogs/ amino acid analogs
Example: Fig 34.42
AZT, a nucleoside analog of thymidine
Quinolones
-another class of synthetic antimicrobial drugs
-interact with bacterial DNA gyrase, prevent the gyrase from
supercoiling bacterial DNA and packaging DNA in the
bacterial cell
DNA gyrase is found in all Bacteria
-fluoroquinolones (ciprofloxacin) are effective for treating both
gram-positive and gram-negative bacterial infections
-broad spectrum
- (over)use in beef and poultry
prevention and treatment of respiratory diseases
Figure 27.18
Ciprofloxacin- urinary tract infections in humans
-treat infections from penicillin-resistant strains of
Bacillus anthracis (bioterrorism agent)
¥What is selective toxicity?
¥Distinguish synthetic chemotherapeutic agents from antiseptics and disinfectants.
¥Describe the action of a growth factor analog.
27.7 Naturally Occurring Antimicrobial Drugs: Antibiotics
Antibiotics: natural antimicrobial compounds produced
by fungi and bacteria (other microorganisms) that inhibit or
kill other microorganisms
-thousands known- <1% are clinically useful
-semisynthetic antibiotics
natural antibiotics structurally modified in the laboratory
?enhanced efficacy?
Antibiotics and Selective Antimicrobial Toxicity
Figure 27.12
The susceptibility of microorganisms to individual antibiotics and
other antimicrobial agents varies significantly
-gram-positive Bacteria and gram-negative Bacteria may differ in
their susceptibility to an individual antibiotic
-broad-spectrum antibiotics are effective on both groups.
broad-spectrum antibiotics may find wider medical
use than a narrow-spectrum antibiotic
(but vancomycin (MRSA) and isoniazid are very useful)
Antibiotics Affecting Protein Synthesis Figure 27.13
Antibiotics inhibit protein synthesis by interacting with the ribosome -
medical and research uses
-streptomycin inhibits protein chain initiation
-tetracycline inhibits protein elongation
Selectivity
-tetracycline is specific for ribosomes of Bacteria
Problem:
antibiotics that inhibit protein synthesis in Bacteria also inhibit protein
synthesis in mitochondria
-tetracycline is still useful because the eukaryotic mitochondria are not
affected at the concentrations used for antimicrobial therapy
-Distinguish antibiotics from growth factor analogs.
- What is a broad-spectrum antibiotic?
- Identify the potential target sites for antibiotics that
inhibit protein synthesis and transcription.
27.8 Beta-lactam Antibiotics
penicillins, cephalosporins, and cephamycins
Figure 27.14 Penicillins and cephalosporins account for 54% of
antibiotics produced and used worldwide.
Figure 27.19 Penicillins, cephalosporins, and cephamycins
-medically important antibiotics, share a structural
component, the beta-lactam ring
Penicillins
-1929 Alexander Fleming characterized an antibacterial product of
the fungus Penicillium chrysogenum as the antibiotic penicillin G
-the first clinically effective antibiotic discovered
-1939 Howard Florey et al. - process for large-scale production
of penicillin
Penicillin G is active primarily against gram-positive Bacteria
-Staphylococcus, Streptococcus pneumoniae, S. pyogenes
- gram-negative Bacteria are impermeable to the antibiotic
Modifications of the penicillin G structure changes properties of
the resulting analogs F 27.19
-semisynthetic penicillins -ampicillin and carbenicillin- are
effective against some gram-negative Bacteria
-modified N-acyl groups of these semisynthetic penicillins allow
them to be transported across gram-negative outer membranes
-resistance: penicillin G is sensitive to beta-lactamase
produced by some penicillin-resistant Bacteria
-semisynthetic oxacillin and methicillin are resistant to
beta-lactamase
Mechanism of Action
Beta-lactam antibiotics inhibit cell wall synthesis, therefore are highly selective
-transpeptidation reaction cross-links two glycan-linked peptide chains (Figure 6.7a).
-transpeptidase enzymes [penicillin binding proteins (PBPs)] bind beta-lactam antibiotics instead of bacterial glycopeptides
Result:
no cross-linking, cell wall continues to be formed, resulting in a weakened, self-degrading cell wall
-osmotic pressure (high inside, low outside) and endogenous autolysins inside the cell, lyse the cell
-bacteriolytic, bacteriocidal
¥Draw the structure of the beta-lactam ring and indicate the site of beta-lactamase activity.
¥How do the beta-lactam antibiotics function?
27.9 Antibiotics from Prokaryotes
Macrolide Antibiotics
Erythromycin (produced by Streptomyces erythreus) contains
large lactone rings connected to sugar molecules (Figure 27.22)
-variations in macrolide ring and sugars = variety of macrolides
-protein synthesis inhibitor binds the 50S subunit of the
ribosome
-use- in place of penicillin in patients allergic to penicillin
or other beta-lactam antibiotics
Tetracyclines Figure 27.23
-produced by Streptomyces
-early broad-spectrum antibiotics, inhibiting almost all
gram-positive and gram-negative Bacteria
-naphthacene ring substituted at several positions to form new
tetracycline analogs
e.g. chlortetracycline has a chlorine atom,
oxytetracycline has a hydroxyl (OH) group, no chlorine
-natural products
-semisynthetic variations are constantly developed
-protein synthesis inhibitor
interferes with 30S ribosomal subunit function
-tetracyclines (and beta-lactam antibiotics) are the most
important groups of antibiotics in clinical use
-also used in veterinary medicine
-in some countries tetracyclines are used as nutritional
supplements for poultry and swine
-this prophylactic veterinary use is now discouraged
-fosters drug resistance
Fig 27.25 Platensimycin
Streptomyces platensis
Inhibits bacterial lipid biosynthesis
Broad spectrum
MRSA, VRE
No toxicity
No resistance
¥What are the biological sources of tetracyclines? Macrolides? Platensimycin?
¥Compare to the sources of penicillin beta-lactam antibiotics.
¥How does each antibiotic lead to death of the affected cell?
IV Control of Viruses
27.10 Antiviral Drugs
Viruses use eukaryotic hosts to reproduce and perform
metabolic functions
-many drugs that control viruses target host structures and
are also toxic for the host
Strategy
- several agents are more toxic for viruses than for the host
-a few chemical agents specifically target viruses
HIV
Antiviral Chemotherapeutic Agents (Table 27.5).
Nucleoside analogs, some of which are
nucleoside reverse transcriptase inhibitors (NRTI),
all work by the same mechanism:
inhibit elongation of the viral nucleic acid polymer (DNA or RNA)
Model compound
-azidothymidine (AZT)
Fig 34.42 AZT inhibits retroviruses -HIV
inhibits RNA-dependent DNA polymerase
-analog of thymidine, substitutes 3ÕN3 for the 3ÕOH in deoxyribose, stops chain elongation
AZT inhibits multiplication of retroviruses by blocking reverse transcription and production of the virally encoded DNA intermediate
Side effects
-normal cell nucleic acid replication is targeted,
causing host toxicity (all of the virus inhibitor drugs)
-NRTIs lose potency with emergence of drug resistance
Table 27.5
-Nevirapine, a non-nucleoside reverse transcriptase inhibitor
(NNRTI), binds to reverse transcriptase
-inhibits reverse transcription
Phosphonoformic acid - inorganic pyrophosphate analog inhibits
internucleotide linkages, preventing synthesis of viral nucleic acids
protease inhibitors (PI) (Figure 27.31) prevent viral replication by
binding the active site of HIV protease, inhibiting processing of
viral polypeptides and virus maturation-less toxicity
fusion inhibitor -enfuvirtide -a 36-amino acid synthetic peptide
-binds gp41 membrane protein of HIV
-stops the conformational changes necessary for the fusion of
viral and target T lymphocyte cell membranes
Influenza Antiviral Agents
-adamantane derivatives - amantadine and rimantadine
synthetic amines that interfere with an influenza A ion transport
protein, inhibiting virus uncoating and replication
-neuraminidase inhibitors - oseltamivir and zanamivir
block the active site of neuraminidase in influenza A and B
viruses, inhibiting virus release from infected cells
Both categories of drugs are used for treatment and prophylaxis
of influenza infection (Section 34.9).
¥Why are there relatively few effective antiviral chemotherapeutic agents? Why aren't such agents used to treat common viral illnesses such as colds?
¥What steps in the viral maturation process are inhibited by nucleoside analogs? By protease inhibitors?
27.12 Antimicrobial drug resistance
-the acquired ability of a microorganism to resist the effects
of a chemotherapeutic agent to which it is normally
susceptible
Origins - antibiotic producers are microorganisms,
-antibiotic-producing microorganisms develop (or have)
resistance mechanisms to neutralize or destroy their
own antibiotics (Streptomyces and Penicillium)
-resistance genes may be transferred between and among
(related) microorganisms by genetic exchange
vertical (chromosomal)
horizontal (plasmids)
Resistance Mechanisms (Table 27.7)
(1) The organism may lack the targeted structure. Mycoplsma
-mycoplasmas, lack bacterial cell wall = resistant to penicillins
(2) The organism may be impermeable to the antibiotic
-gram-negative Bacteria are impermeable to penicillin G
(3) The organism may alter the antibiotic to an inactive form
-many staphylococci contain beta-lactamase that cleaves the
beta-lactam ring of many penicillins (Figure 27.27)
Common Modifications - Figure 27.27
(4) The organism may modify the target of the antibiotic
(e.g., mutation in ribosome)
(5) The organism may develop a resistant biochemical pathway
-Bacteria resistant to sulfonamides modify their metabolism to take
up preformed folic acid from the environment, avoiding the need
for the pathway blocked by sulfonamides or the enzyme that
binds PABA acquires a higher affinity for PABA than sulfonamide
(6) The organism may be able to pump out an antibiotic entering
the cell (efflux)
Mechanism of Resistance Mediated by R Plasmids
Drug-resistant bacteria isolated from patients have R (resistance)
plasmids
-R plasmids encode enzymes that inactivate antimicrobial agents
(beta-lactamase inactivates penicillin -Figure 27.27)
-R plasmids encode enzymes that prevent drug uptake
or
-R plasmids encode proteins that actively pump out the drug
-R plasmids may confer multiple antibiotic resistance
-a single R plasmid may contain several different genes,
each encoding a different antibiotic-inactivating enzyme
Origin of Resistance Plasmids
R plasmids existed before the antibiotic era.
-Escherichia coli freeze-dried in 1946 contained a plasmid with
genes conferring resistance to both tetracycline and
streptomycin
-neither antibiotic was used clinically until several years later
-strains carrying R plasmid genes for resistance to
Semisynthetic penicillins existed before the semisynthetic
penicillins had been synthesized.
-R plasmids with antibiotic resistance genes are found in
nonpathogenic soil Bacteria
? selective advantage for major antibiotic-producing organisms
that are normal soil organisms (Streptomyces) ?
Spread of Antimicrobial Drug Resistance
-medical, veterinary, and agricultural antibiotic use provides
selective conditions for the spread of R plasmids and
confers a selective advantage to bacteria containing R plasmids
-Antibiotic resistance is predictable
-an outcome of ÒnaturalÓ selection
-(Over)use of antimicrobial drugs fosters resistance
-Increased clinical use of an antibiotic is paralleled by emerging
resistance to the same antibiotic
Example: Neisseria gonorrhoeae (gonorrhea) resistance
to ciprofloxacin (fluoroquinolone) / ceftriaxone
Figure 27.28c
Overuse
Good news:
Antibiotics are now prescribed at lower levels than in the past
(generally useful in 20% of cases)
-physicians now prescribe about one-third fewer antibiotics for
treatment of childhood infections than they did 10 years ago
Bad news:
-ineffective dose, patient non-compliance, lead to sublethal
doses of antibiotics for short periods of time, selecting for
resistant strains
Overuse
More bad news:
-Antibiotics used in animal feeds as growth-promoters and
prophylactic additives may lead to resistance
-fluoroquinolones have been extensively used for less than 20 yrs
as growth-promoting and prophylactic agents in agriculture AND
-fluoroquinolone-resistant Campylobacter jejuni have emerged as
foodborne pathogens. Why?
? Routine treatment of poultry flocks with fluoroquinolones to
prevent respiratory diseases?
Antibiotic-Resistant Pathogens
Almost all pathogens are resistant to some antimicrobial agents
since widespread use of antimicrobial chemotherapy began in the
1950s (Figure 27.29)
Penicillin and sulfa drugs are not widely used because many
pathogens have acquired some resistance
Some pathogens are resistant to known antimicrobial agents
-vancomycin-resistant Enterococcus faecium (VRE)
-Mycobacterium tuberculosis (XDR TB)
-methicillin-resistant Staphylococcus aureus (MRSA)
-Pseudomonas aeruginosa
-Candida albicans
Steps to prevent resistance
Limited, appropriate use of antibiotics
Combination therapy
Potential reversal of resistance patterns with restricted use
¥Identify the six basic mechanisms of antibiotic resistance among bacteria.
¥Identify the primary sources of antibiotic resistance genes.
¥What practices encourage the development of antibiotic-resistant pathogens?
Brock Biology of Microorganisms
12th Edition
Chapter 28:
Microbial
Interactions with Humans
II
HARMFUL MICROBIAL INTERACTIONS WITH HUMANS
F 28.12
Pathogens are disease-
producing microorganisms
Various pathogen
strategies cause disease
Virulence
the relative ability of a
pathogen to cause disease
Growth in the body
= infection
Model pathogens:
Neisseria gonorrhoeae
Streptococcus
Clostridium
Escherichia coli
Salmonella
Influenza
28.6 Entry of the Pathogen into the Host
Adherence Table 28.3
Pathogens initiate infections at breaks / wounds in skin /
mucous membranes of the respiratory, digestive, or genitourinary
tract - specific adherence to epithelial cells (Figure 28.13)
Examples:
Tissue specificity
Neisseria gonorrhoeae (gonorrhea)
adheres to urogenital epithelia through a surface protein
Opa (opacity associated protein)
Opa binds CD66 on the surface of human epithelial cells.
Species specificity-
H5N1 strain adheres to bird epithelia
not well to humans
Polysaccharides, proteins, or protein-carbohydrate mixtures
are synthesized and secreted by the bacteria.
slime layer - not attached to the bacteria (Fig 28.4b)
capsule (Figures 28.13 and 28.14) a dense, well-defined
polymer coat surrounding the cell
Functions:
1. adherence between other bacteria
2. protect bacteria from host defense / phagocytosis
Vibrio cholerae
No capsule
Escherichia coli
Extensive capsule
Both adhere to villi Fig 28.13
Fig 28.14 Bacillus anthracis Extensive capsule
Fimbriae and pili
-bacterial cell surface protein structures that function in
mobility
and attachment
-Pili of Neisseria
gonorrhoeae attach to urogenital
epithelium
-Fimbriae- strains of
Escherichia coli with fimbriae
(Figure
28.15)
cause urinary tract infections more frequently than
strains lacking fimbriae
Differences:
Type I fimbriae (Escherichia, Klebsiella,
Salmonella, Shigella)
-uniformly
distributed on the surface of cells
enterotoxic strains of E. coli
express fimbrial proteins
called CFA (colonization factor antigens)
adhere specifically to cells in the small intestine
Fimbriae in E. coli Fig 28.15
Pili are generally longer than fimbriae, with fewer pili found
on
the cell surface.
Flagella also
increase adherence to host cells and
boost
virulence.
Invasion -
getting a foothold in the host
-most
pathogens must penetrate the epithelium to initiate
pathogenicity
-attachment
is established and invasion occurs at small breaks or
lesions in the skin or on mucosal surfaces
-attachment
and invasion can be enhanced on mucosal surfaces
if normal flora is altered
How? Example: antimicrobial chemotherapy
illness
Type organism: Streptococcus
pneumoniae
¥How do Opa proteins on Neisseria
gonorrhoeae influence adherence to mucosal
tissues?
¥What is invasion?
¥How does adherence
initiate invasion?
21.7
Colonization and Growth (Infection)
colonization - pathogen multiplies in wounds, breaks,
mucosal
tissues, even blood
Pathogen must find nutrients
and suitable conditions
in host (temperature, pH, and presence or
absence of oxygen)
-soluble
nutrients -sugars, amino acids, organic acids- are limited
-pathogens
able to use complex nutrients (glycogen) are favored
Iron: concentration influences growth
-transferrin and lactoferrin
in animals bind iron tightly
-some
pathogens require iron
-iron salt solution given to an infected animal
increases the virulence of some pathogens
siderophores called aerobactin
from Escherichia coli
(encoded
on Col V plasmid) removes iron bound to host
transferrin
Localized
Growth/ Infection and Spread in the Body
MRSA
Local infection-
boil that may arise from Staphylococcus skin
infections may lead to generalized (systemic)
infection
– spread of the pathogen through the blood and lymph systems
can lead to extensive bacterial growth in tissues
Some of the
organisms are shed into the bloodstream in large
numbers - bacteremia
Widespread systemic
infection may occur – septicemia
¥Why are colonization and growth necessary for the success of most
pathogens?
¥Identify host factors that limit or accelerate colonization and growth
of a microorganism at a local site.
21.8 Virulence
Virulence is the relative
ability of a parasite to cause disease
Measuring
Virulence
virulence
estimates:
LD50 (lethal dose50) = dose of an agent that
kills 50% of the
animals in a test group
Fig 28.16 Virulence quantified
For S. pneumoniae
-hard
to count the few organisms needed for LD50
highly virulent pathogen - way less than 100 organisms
20?
LD50 for Salmonella typhimurium,
a less virulent pathogen,
is much higher than for S. pneumoniae
2000?
The number of cells of Salmonella typhimurium
required to kill
100% is about 10,000 times greater than the number of cells
needed to achieve 100% death with S. pneumoniae.
Attenuation- loss of virulence
in a pathogen occurs because
nonvirulent mutants may grow
faster in vitro
Successive transfers in vitro select such mutants
Attenuated strains are used for production of many viral vaccines
measles, mumps, rubella, some polio vaccines.
BCG (bacille
Calmette-Guerin) and Mycobacterium
tuberculosis
Toxicity and
Invasiveness
Virulence is due to the ability of a pathogen to cause host damage
through toxicity and invasiveness.
Toxicity is the ability of
an organism to cause disease by means
of a preformed toxin that inhibits host cell
function or kills host cells.
Major virulence factor for Clostridium tetani
is tetanus toxin
-moves to distant parts of the body and
initiates
irreversible muscle contraction and often death of the
host.
Virulence in C. tetani is due almost
exclusively to toxicity.
Invasiveness
Ability of an organism to grow in host tissue in such large numbers
that the pathogen inhibits host function (causes
disease).
Major virulence factor for Streptococcus pneumoniae
=
polysaccharide capsule
-
prevents the phagocytosis of
pathogenic strains
Encapsulated S. pneumoniae cause
extensive host damage
because they are highly invasive
-grow in lung tissues in enormous numbers and initiate
-host inflammatory responses that lead to pneumonia
(nonencapsulated strains are
destroyed by phagocytes)
Most pathogens fall between the extreme toxicity of C. tetani
and the extreme invasiveness of S. pneumoniae
Most successful
pathogens use a combination of toxins
and invasiveness
Salmonella,
Streptococcus pyogenes, Staphylococcus
¥Explain
attenuation
toxicity
invasiveness
¥Give examples
III. Virulence
Factors and Toxins
28.9 Virulence Factors (Table 28.4)
virulence factor
-pathogen-produced extracellular protein that
aids in
the establishment and maintenance of disease
Example; streptococci, staphylococci, and certain clostridia
produce hyaluronidase (enzyme)
-promotes spread of organisms in tissues by
breaking down host
hyaluronic acid (intercellular
cement)
-digestion of the intercellular matrix enables these
organisms to spread from an initial site
Fibrin, Clots,
and Virulence
Coagulase (E) produced
by pathogenic
Staphylococcus
aureus
Coagulase causes fibrin (clot) to be deposited on S.
aureus cells
-protects the coated bacteria from attack by host cells
-accounts for the extremely localized nature of many
staphylococcal infections, as in boils and pimples.
Coagulase-positive Staphylococcus aureus
strains are more
virulent than coagulase-negative
strains
Streptokinase (E) fibrinolytic substance produced by
Streptococcus
pyogenes
Streptokinase dissolves clots, allowing S. pyogenes
to move out
of wound and into surrounding tissues
¥What advantage does the pathogen gain by producing enzymes that digest
structural components of host tissues?
¥How can the activity of coagulase work to
enhance the growth of Staphylococcus aureus?
28.10 Exotoxins Table
28.4
Exotoxins -toxic proteins
released extracellularly
Travel from a focus of infection and cause damage at distant sites.
4 categories;
1.Cytolytic
toxins (CT) - enzymatically attack cells,
cause lysis.
2.Enzymes (E)-
break down host tissues or produce protective
structures for the pathogen
3.AB toxins: AB toxins consist of two covalently bonded
subunits
-B subunit binds to a cell surface receptor, allowing the transfer of
the
toxic A subunit across the cell membrane, where it damages
the
cell
4.Superantigen
toxins (SA)
stimulates large numbers of immune cells, resulting in
extensive
inflammation
Fig 28.18 Cytolytic Toxins--hemolysins
(a)Streptococcus pyogenes - streptolysin
O
(b) Clostridium perfringens- lecithinase
Fig 28.19 Staphylococcal alpha toxin - a cytotoxin
AB toxins
Diphtheria Toxin
(Figure
28.20)
Corynebacterium diphtheriae AB toxin is a
single polypeptide
-fragment B binds a host cell receptor
binding induces
proteolytic cleavage, separating fragments
A
and B
-A enters host cytoplasm - disrupts protein synthesis by
blocking amino
acid transfer from tRNA to the growing
polypeptide chain
-A specifically inactivates elongation factor 2 in eukaryotic
cells
(involved in growth of the polypeptide chain) by
catalyzing
the attachment of adenosine diphosphate
(ADP) ribose
from NAD+ to EF-2, modifying its ability to bind and transfer
amino acids from tRNA, stopping
protein synthesis
Fig 28.20 Action of diphtheria AB toxin
Neurotoxic exotoxins
Botulinum toxin: AB toxin, botulism, Figure 28.21
Most potent biological toxin known
1
mg of botulinum toxin can kill > 1 million guinea
pigs
1
g = 1 BILLION
Botulism
-bioactive toxin complex binds to presynaptic membranes on
the termini of the stimulatory motor neurons at
the
neuromuscular junction, blocking release of acetylcholine
Fig 28.21
Botulinum-poisoned (blocked) muscle
cannot
receive excitatory A signal - contraction is
prevented
Result: flaccid paralysis (death by suffocation)
Botulinum toxin-poisoned (blocked) muscle cannot
receive an
excitatory signal
Contraction is prevented
transmission of the nerve impulse to the muscle through
acetylcholine - muscle receptor binding/excitation
is blocked
Result: flaccid paralysis and death by suffocation
Tetanus toxin AB toxin Figure
28.22
a. Normally,
interneurons release glycine,
an inhibitory
neurotransmitter.
G binds to receptors on the motor neurons,
stopping release of A by motor neurons and inhibiting
muscle
contraction, allowing relaxation.
b. Tetanus AB toxin moves
through motor neurons to the
spinal cord and binds specifically to ganglioside lipids at the
termini of the inhibitory interneurons.
TT blocks G release: motor neurons are not inhibited, leading
to continual release of A and uncontrolled
contraction of the
poisoned muscles.
Uncontrolled contraction results in spastic paralysis
trismus/lockjaw
Fig 28.22
Tetanus -spastic, twitching paralysis
-affected muscles are constantly contracted
Fig 35.21 Tetanus.
Muscles of the mouth- trismus or lockjaw
Respiratory muscles-death due to asphyxiation.
¥Identify features are shared by all exotoxins.
¥Identify the unique features of exotoxins.
¥Are bacterial growth AND infection in the host necessary for the
production of toxins? Explain your answer.
28.11 Enterotoxins
Enterotoxins are exotoxins whose
activity affects the
small intestine
-causes massive secretion of fluid into the
intestinal lumen
-leads to vomiting and diarrhea
Figure 28.23
Cholera toxin action of
cholera toxin
AB enterotoxin produced by Vibrio cholerae
(cholera)
one A subunit : five B subunits
-B subunit binds specifically with the ganglioside
GM1 (a complex
glycolipid) in the cytoplasmic membrane of epithelial cells
-the A subunit crosses the cell membrane,
activates adenyl
cyclase, converts ATP
-> cAMP
-higher cAMP levels
cause secretion of chloride and bicarbonate
ions from mucosal cells into the intestinal lumen (out)
-
secretion of large amounts of water into the lumen
(out)
-
rate of water loss into the small intestine can be
greater
than the reabsorption of water by
the large intestine
-
massive net fluid loss occurs - vomiting, diarrhea
Victims generally die from dehydration/ electrolyte imbalance
Treatment - oral fluid replacement with solutions containing
electrolytes and other solutes
Other enterotoxins
Some enterotoxigenic Escherichia and Salmonella
toxins are
functionally and structurally, related to cholera toxin
-produced
in the gut by colonizing bacteria
-Staphylococcus aureus enterotoxin is a superantigen
-stimulate large numbers of immune lymphocytes
-causes systemic and intestinal inflammatory responses
Shiga toxins are protein synthesis inhibiting AB toxins
Shigella
dysenteriae
E. coli
O157:H7 (Shiga-like toxin)
Foodborne diseases
Food poisoning - acquired by the ingestion of preformed toxin
-growth in host is unnecessary
Staph
enterotoxin, botulism (latency - hours)
Food infections - require active growth to form toxins
V.
cholerae, E.coli,
Salmonella
Food
infections require
bacterial growth in the host,
followed by toxin production (latency - days)
¥What key features are shared by all enterotoxins?
¥Describe the action of Vibrio
cholerae toxin on the small intestine. Why does this
lead to massive fluid loss?
ORT – Oral rehyderation therapy
ORS- oral rehydration
solution
UNICEF /WHO contents of reduced osmolarity
ORS packets 2006.
A 1-liter preparation of ORT solution contains:
sodium chloride (NaCl) - 2.6g
trisodium citrate dehydrate -
2.9g
potassium chloride (KCl) -
1.5g
anhydrous glucose
- 13.5g
recommendation for zinc supplementation
Sports drinks
are formulated to rehydrate healthy individuals
-too
much sugar and too little electrolytes for ORT
28.12 Endotoxins
Endotoxin: the lipopolysaccharide (LPS) produced by
gram-negative Bacteria as part of the outer layer of
cell envelope is toxic
LPS endotoxins are cell-bound and released in
large
amounts only when cells lyse
-in contrast, exotoxins are
secreted products of living cells
(Table 28.5).
Endotoxins have been studied primarily in Escherichia,
Shigella,
and especially Salmonella (gram- Bacteria)
Endotoxin Structure and Function
LPS consists of three covalently linked subunits (Figure 4.23)
lipid A, a core polysaccharide, and the O-polysaccharide.
Fever - universal
symptom
endotoxin stimulates host cells to
release endogenous
pyrogens (cytokines) that
affect temperature control in the brain
Other symptoms
-diarrhea
-rapid decrease in lymphocyte, leukocyte, and
platelet numbers
-release of cytokines leading to general
inflammation
Large doses:
death from hemorrhagic shock and tissue necrosis
Toxicity of endotoxins is MUCH lower than
that of exotoxins (mice)
LD50 for endotoxin is 200-400 mg
per animal
LD50 for botulinum toxin is about
25 pg
about 10,000 fold less!
¥Why do gram-positive Bacteria not produce endotoxins?
¥Why are drug preparations tested for endotoxin?
(Parenteral administration)
Start here 11/5
IV. Host Factors
in Infection
Uncontrollable risk
factors: age, genetic makeup,
tissue specificity
Controllable risk factors: diet, stress
Host Risk
Factors for Infection
Age, Stress, and
Diet
Age -not controllable-
Infectious
diseases are more common in the very
young (<1 yr) and in the very old ( > 50, 65)
Infant diarrhea
pathogens have a greater opportunity to become
established and produce disease
(limited by establishment of local flora? Immunity?)
diarrhea from pathogenic strains of Escherichia coli
is more
frequent in infants <1 year.
Infant botulism
intestinal infection with Clostridium botulinum
- infants <1 yr
C.
botulinum colonizes and
grows, secretes botulinum toxin,
leading to flaccid paralysis
contracted by ingestion of C. botulinum
from soil, air,
(raw honey)
prevented by establishment of the (competitive) intestinal
normal flora in older children and adults
> 50, 65 years
infectious diseases are much more common
much more susceptible to respiratory infections,
particularly influenza
?declining immune response to pathogens?
Stress
(controllable)
fatigue, exertion, poor diet, dehydration, or drastic
climate
changes are stressors
-increase the incidence and severity of infections
Experimental Salmonella infections:
Rats subjected to Salmonella challenge after intense physical
activity
(long periods) show a higher morbidity and mortality
than
rested animals
Hormone studies
- stress hormone cortisone produced at
higher level under stress
Activates
"fight or flee" response
anti-inflammatory agent
-
suppresses inflammation, inhibits activation of
immune
response, promotes infection
Diet (controllable)
Vibrio cholerae numbers necessary to produce cholera in an
exposed individual are drastically reduced if the
individual is malnourished
Dietary
restriction of sucrose and good oral hygiene
eliminate
tooth decay.
Without dietary sucrose, Streptococcus mutans
and
S. sobrinus are unable to
synthesize the polysaccharide
slime layer necessary for adherence to tooth
surfaces.
The Compromised
Host
Hosts in which one or more resistance mechanisms are inactive
-probability of infection is therefore increased.
Healthcare-associated
infections
morbidity -2 million cases / year
mortality -100,000 deaths / year
Hospital procedures - catheterization, injection, surgery etc.,
may introduce microorganisms
Organ transplant patients are treated with immunosuppressive
drugs that reduce patient resistance to infection
General stress of illness
Compromised hosts outside the hospital result from:
smoking, excess consumption of alcohol, i.v.
drug use,
lack of sleep, poor nutrition, acute or chronic infection
with
another agent
Chronic infection example:
HIV infection
HIV-AIDS deaths are generally due to opportunistic pathogens,
microorganisms that do not ordinarily cause disease in an
uncompromised host
Genetic diseases that attack the immune system predispose
individuals to infections - die from infection. Which organisms?
¥Identify factors that control susceptibility to
infection and cannot be controlled by the host.
¥Identify factors that control susceptibility to
infection and can be controlled by the host.
28.14 Innate
Resistance to Infection
Natural Host
Resistance
Examples of species differences
Rabies
Raccoons and skunks are very susceptible to rabies infection.
Opossums rarely acquire rabies.
Anthrax (Bacillus
anthracis)
-birds are totally resistant to anthrax
-fatal blood poisoning in cattle
-
cutaneous anthrax -
mild pustules in humans
-pulmonary, or airborne, anthrax (bioterrorism) is
>90% fatal in humans
HIV infection occurs
only in higher-order primates
(great apes and humans)
Why?
CXCR4
protein on T cells and
CCR5
protein on macrophages
are uniquely
expressed in primates
They act as cell surface receptors and specifically interact with
HIV
gp120 protein
Other animals lack these receptors, cannot bind HIV,
and are thus protected from HIV infection
Figure 28.25
Physical and Chemical Defenses of the Body
Skin - effective
barrier to the penetration of microorganisms.
Sebaceous
glands in the skin secrete fatty acids and lactic
acid, lowering to pH 5
-inhibits colonization of many pathogenic bacteria
(blood and
internal organs are about pH 7.4)
Ciliated epithelial
cells on nasopharynx and trachea move cells into
oral secretions
-expectorated or swallowed and killed in the stomach
-continual movement also prevents adherence
Stomach acidity (pH 2) kills/ reduces numbers of ingested
bacteria
Resident microflora and pH prevent
colonization
stomach (pH 2)
small intestine (pH 5) and
large intestine (pH 6–7) (bacterial numbers of 1010
or more
per gram of contents)
Lysozyme found in lumen of the kidney and the
surface of the eye
Tissue
Specificity (Table 28.6) Pathogens must be able to
adhere and colonize at the site of exposure
Adherence to an exposure site does not guarantee colonization:
The site must meet nutritional and metabolic requirements of the
Pathogen
Pathogens cause disease at tissue-specific sites:
Clostridium tetani cells or endospores
introduced into a deep
wound promotes growth and tetanus toxin production
in the anoxic zones created by local tissue death
C.tetani pathogen is killed by the acidity of the stomach
Enteric bacteria - Salmonella and Shigella
-colonize the intestinal tract, cause food infections
-do not cause wound infections
- Identify physical and chemical barriers to pathogens.
How
might these barriers be compromised?
- How might preexisting infection compromise an
otherwise healthy host?