Transcript (F`).

Medical Microbiology
Bacterial Genetics
You have known…
Science of genetics describes and analyze heredity of physiologic
functions that form the properties of organism.
These properties are determined by the total of all the genetic
information named genome.
Basic unit of genetics is gene, a segment of DNA that carries in its
nucleotide sequence information for a specific biochemical or
physiologic property.
A gene is relatively stable but occasionally may undergo a
nucleotide change, such a change is called as mutation.
Mutations may occur spontaneously or can be induced by a number
of physical or chemical agents.
Bacteria may have changes including:
 Morphological and/or structural changes
 Variations of cultural characteristics and
biochemical reactions
 Changes in virulence
 Variation in antigenicity
 Changes in drug resistance
These variations can be divided into two types:
▲ Phenotypic variation: non-heritable
▲ Genotypic variation: heritable (mutation)
Bacterial Genome
DNA/Genome:
Genetic materials relative to bacterial mutation.
A. chromosome
B. out of chromosome: a) plasmid
b) phage
c) transposable genetic element
1. Chromosomal DNA
Bacterial chromosome consists of a single,
circle of double-stranded DNA.
▲ 2 mm long in average
▲ Usually < 5 Mb (1 Mb = 1024 Kb)
2. Plasmid
Transfer property
▲ Conjugative plasmid：mediate conjugation through sex pilus
▲ Non-conjugative plasmid: can not mediate conjugation
because of no gene for encoding sex pilus
Phenotypic effect
▲ Fertility plasmid (F factor): has a sex pilus-encoding gene
▲ Resistance plasmid (R factors): contains genes that encoding
enzymes to destroy antibiotics
Structure of R Factor
RTF (Resistance Transfer Factor)
▲ Conjugative plasmid
RTF
▲ Transfer genes
R determinant
▲ Resistance genes
▲ Transposons (Tn, 转座子)
R determinant
3. Phage
Phages are obligate intracellular
parasites that specially propagate
in bacteria (bacterial virus).
▲ Nucleic acid: DNA (dsDNA or
ssDNA) or RNA (dsRNA or ss RNA).
▲ Protein: function in infection
and protect nucleic acid.
Structure of T4 phage
Capsid
DNA
Contractile
Sheath
Head
Tail
Tail Fibers
Base Plate
▲ Head: DNA or RNA+protein coat (capsid)
▲ Tail: is composed of a hollow core surrounded by a sheath
with base plate and at the end.
Interaction between phages and bacteria
Phages are wide spread in nature but usually has a specificity of
host bacteria.
Infection with a virulent phage results in phage replication with
the production of new phage particles and their subsequent
release causes death of host bacteria.
adsorption
penetration
biosynthesis
maturation / release
Infection with a temperate phage does not necessarily lead to
bacterial death, but phage’s nucleic acid integrates into
bacterial chromosome without new phase production.
adsorption
penetration
integration
Virulent phage
I. Adsorption
Recognition of host bacterial
surface receptors by tail fibers
II. Penetration
Phage’s nucleic acid is
injected through hollow
core
into
bacterial
cytoplasm.
Virulent phage
III. Biosynthesis
IV. Maturation and Release
Temperate Phage (Prophage formation)
II. peneration
I. adsorption
III. integrate of phage
DNA into host genome
IV. prophage replicates along with
chromosome of lysogenic bacterium
4. Transposable Genetic Elements
Definition:
segments of DNA that have the capacity to
move from one bacterial DNA molecule (bacterial
chromosome or plasmid) to another or from one
location to another in the same one DNA molecule
(jumping gene / movable gene)
Types of Transposable Genetic Elements
▲ Insertion sequences (IS)
▲ Transposons (Tn)
Insertion sequence (IS)
▲Definition: a type of transposable Genetic Elements only has
a transposase-encoding gene alone.
▲ Structure: a small DNA has reverse repeated sequences at
the two ends that are involved in transposition. In the
middle there is a transposase coding gene.
▲ Function: introduction of an insertion sequence into a
bacterial gene will result in inactivation of the gene.
ABCDEFG
Transposase
GFEDCBA
Transposon (Tn)
▲ Definition: a type of transposable Genetic Elements has both
insertion sequences (IS) and other genes.
▲ Structure: the extra genes are located between the terminal
repeated sequences.
▲ Function: Since transposons can jump from one DNA molecule
to another and frequently carry antibiotic-resistant genes, the
transposons mediate drug resistance in bacteria.
IS
Resistance Gene(s)
IS
IS
Resistance Gene(s)
IS
Types of Mutation
Self Mutations: Spontaneously occur with low frequency.
Gene transfer and recombination: high frequency.
One bacterium (recipient) uptake exogenous DNA
segment from another bacterium (donor) or phage (Gene
transfer) and then the DNA segment is incorporated into
DNA of itself (recombination).
Major mechanisms of bacterial gene transfer
▲ Transformation
▲ Transduction
▲ Conjugation
▲ Lysogenic conversion
Transformation
Definition: a bacterial
recipient uptake naked
DNA segment offered by
bacterial
donor
in
environment and then
the
DNA
segment
recombinate with the
recipient’s chromosomal
DNA.
Significance for transformation
Transformation occurs in nature and it can lead to
increased virulence ( e.g. S. pneumoniae) and drug
resistance.
Transduction
Definition: a bacterial donor’s chromosomal DNA segment
transferred to a bacterial recipient by way of a phage, and
then the DNA segment recombinate with the recipient’s
chromosomal DNA .
▲ Generalized Transduction: incorrect assemblage (any genes).
▲ Specialized transduction: Sometimes, during activation of
prophage, the excised
segments at its ends.
phage’s DNA contains host DNA
Lysogenic conversion
Definition: a bacterial recipient is infected with
phage from a bacterial donor, and the phage’s
genes recombinate with recipient’s chromosomal
DNA.
As an example, Corynebacterium diphtheriae will
produces diphtheria toxin after it is infected by the
β- phage, because the gene encoding the toxin is
carried by the phage.
Conjugation
Definition: Gene transfer from a
donor to a recipient by direct
contact between two bacterial
Donor
cells through sex pili.
▲ Donor: a bacterium with F factor can
produce sex pili.
▲ Recipient: a F factor-absent bacterium.
Recipient
F+ factor-depecdent Conjugation
F+
F+
F-
F+
F+
F-
F+
F+
F-
F+
The F+ bacterium transfers extra chain of F+ factor and then
the completed F+ factors in the two bacteria is synthesized
by rolling circle replication.
High-frequency recombination (Hfr)
F factor is integrated into bacterial chromosome.
Bacterial DNA is transferred with a high frequency.
F factor is difficulty to be transferred.
Hfr
Hfr
F-
Hfr
F-
F-
Hfr
F-
F’ factor-depecdent Conjugation
F factor in bacterial DNA is excised but it carries
bacterial chromosomal DNA at its two sides (F’).
Hfr
F’
F’
F-
F’
F’
Summary
The most important contents in this lecture are
displayed as the followings:
1) The Agents (plasmid, phage, bacterial chromosomal DNA)
associated with bacterial mutation.
2) Concepts of Transformation, Transduction, Conjugation
and Lysogenic conversion.
3) The Significance of bacterial mutation (e.g. bacterial
virulence, drug resistance and antigenicity).
Medical Microbiology
Bacterial Infection
Bacterial Pathogenicity
Terms :
Pathogen: disease-causing microbe.
Opportunistic pathogen: A microbe does not cause
diseases in normal conditions, but can cause diseases
in some certain conditions.
Pathogenicity: ability of a microbe to cause diseases.
Virulence: quantitative ability of a microbe to cause
disease (invasiveness and toxigenicity).
LD50: the number of pathogen required to cause death
in half of the exposed hosts.
Source of infection
Exogenous infection
Infectious agents come from environment or hosts
(patient, diseased animal or carrier).
▲ carrier: individuals (human and animal) infected
with microbes but no clinical signs or symptoms.
Endogenous infection
Normal flora act as infections agents under some
certain conditions (opportunistic infection).
Normal flora
Microbial populations inhabit on skin and
mucosa of healthy normal persons.
▲ Gastrointestinal tract
▲ Urogenital tract
▲ Skin and Conjunctiva (结膜)
Physiological role of normal flora
▲ Antagonism: a) biolfilm; b) antimicrobial agents.
▲ Trophism: a) digestion of foods; b) production of
vitamins (K and B).
▲ Immunoenhancement: promotes development of
mucosal immune system.
▲ Anti-tumorigenesis and anti-apolexis(衰老): e.g.
eliminate nitrite and anti-oxidation (SOD).
Opportunistic infections
The conditions required by normal
flora to cause diseases.
▲ Translocation of normal flora.
▲ Suppression of normal flora.
▲ Low immunity of human body.
Hospital acquired infections
(Nosocomial infections)
New infections after 48 hours of hospital
admission.
Pathogenic process of bacteria
Generally, infection process caused by a bacterial
pathogen involves the four steps as the following:
▲Adhesion (chemotaxis)
▲ Survival / propagation
▲ Penetration and spread
▲ Tissue injury
Virulence: adhesins, invasive enzymes and toxins.
1. Adhesion
A process of recognition and combination of
microbial adhesins with receptors on the
surface of host cells
▲ lipoteichoic acid (G+ bacteria)
▲ ordinary pilus and outer membrane proteins (G- bacteria)
BACTERIUM
adhesin
receptor
EPITHELIUM
2. Survival / propagation
•Anti-phagocytosis: e.g. capsules.
•propagation: probably due to stress.
3. Penetration and Spread
▲ Collagenase: hydrolyze collagens.
▲ Hyaluronidas: hydrolyze hyaluronic acid.
▲ Coagulase: agglutinate fibrinogen.
▲ Streptokinase/fibrinolysin: the former activates fibrinogenase
to thrombin, and the latter directly hydrolyze fibrin.
▲ Cytolysins: 1) hemolysin (to lyse erythrocyte or tissue cells )
2) Leukocisin (to kill leukocyte or tissue cells )
4. Tissue injury
▲ Exotoxin
▲ Endotoxin
▲ Immunopathological reaction
4.1. Exotoxin
▲ Cytotoxin: e.g. diphtheria toxin
▲ Nuerotoxin: e.g. tetanospasmin
▲ Enterotoxin: e.g. cholera enterotoxin
diphtheria toxin
4.1. Exotoxin
tetanospasmin
cholera
enterotoxin
A-B type exotoxins
▲ Subunit A is toxic, while subunit B is the ligand of
cell’s receptor to mediate A subunit into host cells.
Cell surface
Toxic
Binding
A
B
4.2. Endotoxin (LPS)
▲Fever: a typical pyrogen.
▲Leukocytoreaction: leukopenia/leukocytosis.
▲DIC: disseminated intravascular coagulation.
▲ Shock: dilatation of small blood vessels
▲ Inflammation: IL-1β , TNF-αand IL-6.
LPS
Major difference between endotoxin and exotoxin
Property
Endotoxin
Exotoxin
Chemical nature
Lipopolysaccharide
( MW = 800-1, 000 kDa )
Protein or peptide
( MW = 50-1, 000 kDa )
Relationship to bacteria
Part of outer membrane
Extracellular
Denatured by boiling
No
Usually
Antigenicity
Yes
Yes
Form toxoid
No
Yes
Toxicity
Relatively low
( > 100 µg )
Relatively high
( approximate 1 µg )
Pyrogenicity
Yes
Occasionally
Specificity to host cells
Low degree
High degree
Enzymatic activity
No
Usually
4.3. Immunopathological reaction
Human immune responses to bacteria may
cause tissue injury by:
1. Numerous cytokines and complement activation.
2. Continuously generated bacterial antigens will subsequently
causet humoral antibodies and cell mediated immunity, which
resulting in chronic immunopathological injury.
3. Some of bacterial antigens (e.g. M protein of Streptococcus)
react with host tissue antigens to cause autoimmunity.
Number and route
▲ Invaded mumber of bacteria:
Different pathogens need different bacterial
number to cause diseases.
▲ Suitable invading route of bacteria:
Most of bacteria require suitablec invading routes
to cause diseases (e.g. Clostridum tetani causes
disease through wounds and Mycobacterium
tuberculosis has multiple invading routes).
Types of infection
According to infectious state:
▲ Inapparent or subclinical infection: The infection
with
no
manifesting
clinical
signs
and
symptoms.
▲ Latent infection: The infection is inactive but
maintain potential to cause diseases.
▲ Apparent infection: clinical signs and symptoms.
According to infectious site:
▲ Local infection; ▲ Systemic infection
Systemic infection
▲ Bacteremia : Bacteria enter bloodstream
without propagation in bloodstream.
▲ Toxemia: Exotoxin or endotoxin rather than
bacteria enters bloodstream.
▲ Septicemia : Bacteria enter bloodstream with
propagation and release virulent factors.
▲ Pyemia : Pyogenic bacteria enter bloodstream
with propagation and release virulent factors,
and then spread through bloodstream into the
target organs to form pyogenic foci.
Summary
The most important contents in this lecture are displayed as the
followings:
1) Concepts of virulence, normal flora, hospital acquired
infection, latent infection, toxemia, septicemia, endotoxemia
and pyemia.
2) The physiologic role of normal flora.
3) The conditions for generation of opportunistic infection.
4) The difference between exotoxin and endotoxin.
5) The pathogenic effects of endotoxin.
Medical Microbiology
Anti-infection Immunity
Innate Immunity
Phagocytosis: mononuclear-macrophages,
neutrophils.
Complements: lyse bacteria
Others: lysozyme, antibacterial peptide.
Acquired Immunity
Antibody: antibacterial antibody and
antitoxin (IgM, IgG and SIgA).
T lymphocyte: cytotoxicity (CD4Th1, CTL).
Medical Microbiology
Laboratory Diagnosis
Diagnostic procedure
▲ Sample: collect fresh sample from f o c u s o f
infection using aseptic technique.
▲Isolated culture: “gold standard”, strains.
▲Etiological agent detection: Bacterial antigens
(ELISA, etc.) and DNA (PCR).
▲Serological examination:early dignosis (IgM).