MEDICAL MICROBIOLOGY

Course for medical students

Associate Professor L. Yocheva, Ph.D.

Department of Biology, Medical Genetics and

Microbiology
Medical Faculty
SU “St. Kliment Ohridski”

Email: [email protected]
 Lecture 2
Bacterial Structures

1. Essential structures of bacterial cell. Cytoplasmic

structures. Cell wall, peptidoglycan.

2. Gram-positive and Gram-negative bacteria.

Bacterial exceptions.

3. External structures.
 Key Words

• Peptidoglycan
• Teichoic acids
• Penicillin-binding proteins (PBSs)
• LPS (O antigens and Endotoxin - Lipid A)
 Some Clinical Terms
• Normally, the blood is sterile
• Exotoxins – released by both Gram-positive and Gram-
negative bacteria from cell to surrounded media. Different
toxins with different effects.
• Endotoxin is lipid A – normal part of outer membrane of
Gram-negative bacteria. It is very toxic and is released
when the bacterial cells undergoes lysis.
• Bacteremia – bacteria are in the blood stream for a short
time (10-15 minutes), no clinical symptoms
• Sepsis - bacteria are in the blood stream, they multiply,
severe clinical symptoms
• Septic shock – bacteria are in the blood, they multiply,
clinic of sepsis and dysfunction of many organs
• Endotoxic shock - caused by Gram-negative bacteria
• Septic shock - caused by fungi and Gram-positive
bacteria.
 Some Clinical Terms
• Serum (sera or serums plural): The clear liquid that can
be separated from clotted blood.
• Diagnostic serum, immune serum, or antiserum - a serum
containing antibodies that are specific for one or more
antigens. Commercially available.
• Meningitis is a inflammation of the meninges (the
membranes), covering the brain and the spinal cord.
• Gas gangrene also called myonecrosis. Wound infection.
• Tetanus – spastic paralysis occurs. Wound infection.
• Botulism (food born infection) with flaccid (atonic)
paralysis.
• Anthrax – the severe infection of the skin, intestines or
lungs. B. anthracis was used for bioterrorism.
 Prokaryotic Cell Structure
• Essential structural components:
- DNA
- ribosomes
- cytoplasm
- cytoplasmic membrane
- cell wall

• Non-essential organelles:
- capsule
- flagellum
- pili
- cytoplasmic inclusions
- spore
- plasmid
Prokaryotic Cell Structure
 Essential (Obligatory)
Structures of Bacterial Cell
 Cytoplasmic Membrane
• Essential structure of bacterial cell
• Keeps the 'inside' of the cell separated from the
'outside'
• Semipermeable phospholipid bilayer, containing
some proteins
• Fluid-mosaic model
• Lack of sterols (with the exception of members
of genus Mycoplasma). Presence of sterol-like
molecules (hopanoids)
• Contains mesosomes
• Target for some of the antibiotics
The Fluid-Mosaic Model of Cell Membrane

Lipid bilayers with

floating proteins:
– amphipathic lipids
• specific orientation
• polar ends
(hydrophilic –
interact with water)
• non-polar tails
(hydrophobic –
insoluble in water)
– membrane proteins
• peripheral
• integral
The Fluid-Mosaic Model of Cell Membrane

Membrane proteins
• transporters - in
facilitated diffusion
and active transport
• porins - chanels
• enzimes
 Mesosomes

• Folded invaginations in the plasma

membrane
• The function of mesosomes is not fully
understood
• Mesosomes are associated with:
– DNA replication and septum
formation
– Cellular
respiration
Electron Transport
 Cytoplasmic Membrane Functions
1. Selective permeability (active transport and
facilitated diffusion)
2. Electron transport and oxidative
phosphorylation of aerobic bacteria (as the
analog of mitochondria)
3. Excretion of toxins and other waste products
4. Biosynthetic function – enzymes, transporters
5. Interacts with external environment - receptors for
detection of and response to chemicals in
surroundings
6. Involved in the cell division and DNA replication
 Cytoplasm

• Cytoplasm is the water-like fluid inside

the bacterial cell

• The cytoplasm contains the DNA

chromosome, messenger RNA (mRNA),
ribosomes, proteins, enzymes,
metabolites, etc.
 Nucleoid
• Essential structure
• Bacterial analog of nucleus
• Bacterial chromosome is a single, double-
stranded, circular DNA, supercoiled
• Some bacteria have linear chromosome
• No nuclear membrane, no histones
• Complex structure: DNA, nucleoid proteins aid
in folding and RNA
• Target for some antibiotics
Nucleoid
 Ribosomes
• Ribosomes are essential cellular structures
• Complex protein/rRNA structures
• Smaller than eukaryotic ribosomes (30S + 50S = 70S)
• Bacterial ribosomal RNA
– 16S small subunit – unique sequence
– 23S and 5S in large subunit
• Place of translation and protein
synthesis (polyribosomes)
• Ribosomes are important target of
some antibiotics

polyribosomes
(polysomes)
 Cell Envelope

• Covers bacterial cells and protects them

• Composed of a cell membrane and a cell

wall

• Bacterial cell wall - unique to prokaryotes

 Bacterial Cell Wall
• Essential structure
• Rigid structure, surrounding the cytoplasmic
membrane
• Provides protection
• Determines the shape
• Resistant to osmotic pressure in the cell
• May contribute to pathogenicity
• Contains peptidoglycan
• Determines the differences between Gram-
positive and Gram-negative bacteria
• Exception: bacteria of the genus Mycoplasma do
NOT contain a cell wall and peptidoglycan
 Peptidoglycan

• Structural component of the cell wall

• Heteropolymer with a three-dimensional
structure
• 2-40% of the dry weight of the cell
• Unique to all bacteria
• Gram positive and Gram negative bacteria
contain peptidoglycan
 Peptidoglycan
Peptidoglycan (also known as murein, mucopeptide)
consists of sugars and peptides that forms a murein
sac - mesh-like layer outside the plasma membrane.
 Carbohydrate Chains

• They are linear and identical for all bacteria

• Each strand is built of two alternating sugars:
- N-acetylglucosamine (NAG)
- N-acetylmuramic acid (NAM)
• Each chain contains 20 to 100 of them
• The two types of sugars are connected by
β-(1,4) glycosidic bond
• The β-(1,4) linkage is cleaving by lysozyme
• The result is destruction of peptidoglycan (PG)
Lysozyme Activity

β-(1,4) glycosidic
bond

Lysozyme
 Peptide Chains

1. Side chains - tetrapeptides always attached

to the N-acetylmuramic acid (NAM).
• There are some variations in the composition
of side chains in different bacterial species.

2. Transverse peptide bonds or bridges -

they connect two adjacent side chains

• The final result is cross linking of

carbohydrate chains
Structure of Peptidoglycan

Peptide bridge
or bond
Structure of Peptidoglycan
Variations in Composition of Peptidoglycan
 Peptidoglycan Components Unique
to the Bacteria

• N-acetylmuramic acid

• D-amino acids: D-Glutamine and D-Alanine

• Diaminopimelic acid, a precursor of L-Lysine,

characteristic of some Gram-negative bacteria
 Stages in Peptidoglycan Synthesis.
Antibiotics, that Act at this Levels
Three Stages in Peptidoglycan Synthesis
1. Cytoplasm stage. Disaccharide pentapeptide is
formed. Antibiotics are Fosfomycin and
Cycloserine
2. Membrane stage. Bactoprenol is a transporter of
disaccharide pentapeptide. Antibiotic is Bacitracin
3. Cell wall stage. Disaccharide pentapeptide is
attached to peptidoglycan (transpeptidation). The
pentapeptide is converted to a tetrapeptide.
Antibiotics, that act at this level are Penicillin and
Vancomycin
 Peptidoglycan State

• Peptidoglycan is synthesized and

degraded permanently

• This is done with the aid of enzymes

for synthesis or respectively enzymes
for degradation
 Enzymes for Synthesis

• Many enzymes such as transpeptidases and

carboxypeptidases are involved in the synthesis
of PG at the cell wall level

• Transpeptidases and carboxypeptidases are

called penicillin-binding proteins (PBPs)

• They are located over the cell membrane

 Enzymes That Can Degrade the PG

• Glycosidases - degrade the carbohydrate

chains

• Amidases - destroy tetrapeptides

• Endopeptidases - destroy cross bridges

created in transpeptidation
Enzymes for Degradation of
Peptidoglycan
 Clinical Importance of PG

• Peptidoglycan is a target for the action of

many antibiotics

• Peptidoglycan is a target for the action of

the enzyme lysozyme

• Disruption of peptidoglycan leads to

rapid death of the bacterial cell
 Gram-Positive Cell Wall Components
1. Multiple layers of PG:
• Form very thick, rigid cell walls
• PG constitutes about 40% of the dry weight of the
bacterial cell
• Peptidoglycan layers are up to 60
• PG is sufficiently porous to allow diffusion of
different small molecules to the plasma
membrane
2. Teichoic and lipoteichoic acids:
• Found only in Gram-positive bacteria
• Ribitolphosphate or glycerolphosphate to which
are attached sugars and D-alanine
3. Some proteins and carbohydrates
Teichoic and Lipoteichoic Acids Function

• Teichoic and lipoteichoic acids are

important factors of virulence

• Major surface antigens in Gram+ bacteria

• Lipoteichoic acids make a connection

between the PG and cell membrane

• When fall into the blood stream, teichoic

and lipoteichoic acids can cause fever,
vasodilation and shock
Cell Wall in Gram-Positive Bacteria
Gram-Negative Cell Wall Components

1. Peptidoglycan:
• Located over the cell membrane
• It is only 2% of the dry weight of the
cell and consist of 1-3 layers
2. Outer membrane
3. Periplasmic space:
• Located between the inner and outer
membrane
• Rich with secreted proteins and
important enzymes
 Outer Membrane

• Unique structure for all Gram-negative bacteria

• Virulence factor
• Protective (barrier) function
• Consist of:
– Asymmetric membrane: phospholipid layer
and Lipopolysaccharide (LPS) layer
– Proteins - porins, lipoproteins, etc.
Cell Wall in Gram-Negative Bacteria
 Lipopolysaccharide(LPS)

Consists of three parts:

1. Lipid A or endotoxin - responsible for
the endotoxin activity of LPS
2. The core polysaccharide – a branched
polysaccharide of 9 to 12 sugars
3. The O antigen:
• Attached to the core and extending away from
the bacteria
• It is a long polysaccharide consisting of 50 to 100
repeating saccharide units
• It determines the virulence type (S colonies)
Lipopolysaccharide(LPS)
 Lipopolysaccharide(LPS)

Lipid A Cor
O polysaccharire
toxic part polysaccharide
(endotoxin) R antigen O antigen
 Lipooligosaccharide (LOS)
• Lipooligosaccharide, (LOS) lacks the
O-antigen portion of the LPS
• LOS molecule is more simple and
shorter than LPS
• Neisseria, Heamophilus, Bordetella spp.
etc. are pathogenic bacteria with LOS
• R-antigen (R colonies)

• LOS determine the sensitivity of these

bacteria to some antibiotics and make them
more susceptible to host-mediated defense
 Lipid A (Endotoxin)

• Normal part of outer membrane of all

Gram-negative bacteria

• Released when the bacterial cells

undergoes lysis

• Thermostable (for 2 hours, at 100 ℃)

• It is extremely toxic, have the same toxic

effects in all Gram-negative bacteria
 Effects of Lipid A

• B cell activation (B-cell mitogen)

• Stimulates synthesis of cytokines (IL-1 and
TNFα)
• Pyrogenic effect
• Shwartzman reaction – disseminated
intravascular coagulation
• Endotoxic shock
 Some Proteins of Outer Membrane

• OmpA - structural protein, receptor for

phages
• Porins form porin channels that travers the
membrane and allows passage of some
molecules
• Lipoproteins - connect outer membrane with
the peptidoglycan
 Conclusions About the Cell Wall
1. The Gram-negative outer membrane blocks the
passage of many antibiotics that attack the
peptidoglycan, e.g. Penicillin is unable to pass
through
2. The Gram-positive cell wall is thick, but porous
and low weight molecules like Penicillin can pass
through it to damage the cell membrane or PG
3. Gram-negative cell walls are more complex and
strong than gram-positive walls. They can be
disrupted only by antibiotics for Gram-negative
bacteria
 Bacterial Exceptions

• Bacteria with atypical bacteria cell wall:

the genus Mycobacterium
˗ a cell wall structure with a high content of
lipids, waxes, and fatty acids
˗ Ziehl-Neelsen stain instead of Gram stain.

• Bacteria without cell wall:

the genus Mycoplasma:
˗ the cell membrane is stabilized by sterols
 Bacteria with Atypical Cell Wall

• These cell envelopes resemble Gram positive bacteria in having

polysaccharide covalently bound to the peptidoglycan.
• Unique polysaccharides – arabinogalactans, arabinomannans, lipomannans
• Additional mycolic acids (long, branch chained fatty acids) are covalently
linked to this polysaccharide.
• Other mycolic acid containing compounds and complex lipids form a thick
waxy membranous layer outside the peptidoglycan layer.
• Pathogenic species: Mycobacterium tuberculosis, Nocardia spp., etc.
Bacteria with Impaired Cell Wall - L Forms

• These bacteria are induced by

antibiotic treatment

• Fully resistant to antibiotics

• Can be cultivated

• Can revert to normal form if antibiotic therapy

is interrupted
 L-form of Bacillus subtilis

TEM of L-form Bacillus subtilis.

The cells lack the electron-dense cell wall of normal bacteria.
External and Non-essential
Bacterial Structures
 Fimbriae (or pili)

• Hair like structures on the outside of

bacteria
• Composed of protein (pilin)
• Antigens
• Shorter and thinner than the flagella
• Not used for movement
• Two types:
˗ adhesive fimbriae
˗ sex pili
 Adhesive Fimbriae

• The tips of the fimbriae contain proteins

(adhesins) that bind to specific receptors
on the host cell surfaces.

• Important virulence factor for bacterial

adhesion, colonization and infection.
˗ e.g. Escherichia coli, Neisseria gonorrhoeae,
Bordetella pertussis, etc.
Adhesive Tips of Pili
 Adhesive Fimbriae

• Main function – adhesion

• Present in large number - from 100 to 1000
per cell
 Sex Pili

• Rigid tubular structures, 2 - 4 of them per cell

• Encoded by a F-plasmid
• Function - to connect two bacterial cells for
DNA transfer (conjugation)
• Molecules of DNA pass through the hollow
pilus
• Only bacteria possessing F-plasmid are
able to produce conjugation pili
Sex Pilus
 Flagella
• Provide movement of bacterial cells in liquid
and semisolid media
• Flagella are present in curved bacteria
and in some rods
• Cocci don’t posses flagella
• Part of the flagella is immersed in the cell
wall and the cytoplasmic membrane
• The other part protrudes above the cell as a
long filament
Fimbriae, Sex Pili, Flagelum
Arrangement of Flagella
 Structure of the Flagella

• Basal body, rod and rings (2 or 4). The rings

anchor the flagellum to the cell wall and
plasma membrane
• A hook connects basal body with filament
• Filament: long spiral helical structure with
a central axial channel
• Filament is built from identical protein
subunits called flagellin
• Flagellin is antigen (H antigens)
Structure of the Flagella
 Synthesis of Flagella
• The construction of flagellum is carried out
in stages:
– First is built the basal body
– After that – the hook
– Filament is built last; each subunit flagellin
passes along the axial center channel from
the base to the top

• If the filament is removed, it would

recovered for about 3-6 min
 Flagella Movement

• The movement originated in the basal body and

is an energy dependent process (ATP)

• Transmitted to the hook and to the filament

.
• The filaments rotate at 360° at a high speed

• The flagellar filament reaches 200 to 1000 rpm

• Periodically the direction of rotation is changed

 Movement of Bacterial Cells with Flagella
1. Forward movement, counter-clockwise
rotation of the flagella

2. Clockwise rotation leads to stationary rolling

and spinning (tumble). The result is change of
the direction of movement

3. Again forward movement,

etc. – “random walk”
 Bacterial Chemotaxis
(Attractants and Repellents, Chemoreceptors)
 Axial Filaments

• Some bacteria can move with axial

filament (endoflagella)
• Completely located inside the cell along
its length
• Spirochetes move by axial filaments (have
no flagella)
• Produce movement by:
– Rotation around the longitudinal axis
(corkscrew-motility)
– Folding
– Unfolding
• Axial filaments are composed of
protein, similar to flagellin
 Axial Filaments

Transverse section of spirochete

Mechanism of Movement by Flagella and
Axial Filaments
 Capsules
• Surround many bacterial cells to protect
them
• They are tight fitting
• Most capsules are polysaccharides – e.g. S.
pneumoniae has more than 90 serotypes (each
with different capsular antigens)
• Only a few capsules are polypeptides or
proteins – e.g. B. anthracis
• Antigens – K-antigen
• S. Тyphi have a microcapsule, which is
microcapsular Vi antigen
The Capsules - Non-Essential Organelles

• Some bacteria produce capsules

(encapsulated bacteria), others do
not (strain specific feature)

• The synthesis of capsules depends very

much on growth conditions
 Functions of the Capsules

• The capsules are major virulence factors of

some bacteria.
• The loss of the capsules results in a loss of
the virulence

• The functions of the capsules are:

1. Antiphagocytic effect
2. Adherence and colonization
Phagocytosis of Capsulated and
Non-encapsulated Bacteria
 Methods for Capsules Visualization

Two important test exist to visualize capsules:

1. India ink stain: the stain is not taken by
capsules, they appear as a transparent
halo around the cell.

2. Quellung reaction: the bacteria are mixed

with diagnostic serums. They contain
antibodies that bind to the capsular antigens.
As a result the capsule swells and this can
be visualized microscopically.
India Ink Stain

The capsule appears

as a halo around the
cell (S. pneumoniae)

The background is
stained with India ink
 Quellung reaction or
Neufeld Test for Capsular Swelling

Used as classical
immunological test for
S. pneumoniae serotyping

S. pneumoniae, phase contrast,

Quellung reaction
 Practical Application of the Capsules

1. Capsules have a diagnostic relevance in the

identification of serotypes (serotyping), i.e.
this means to determine different capsular
antigens.
• Some serotypes are pathogenic, others
are not.

2. Capsular polysaccharide antigens are used

for the preparation of some vaccines
Encapsulated Strain of Pseudomonas aeruginosa

М colonies

Pseudomonas aeruginosa may be

surrounded by a thick mucus capsule
 Slime (Glycocalyx)

• Less bound to the cell surface than the

capsules (loose fitting)
• Amorphous material (polysaccharides),
secreted extracellularly by some bacteria
• Freely soluble in water
• Protects cells from drying
• It allows adherence of the cells to different
surfaces and production of biofilm
Slime and Capsule
 Biofilms
• Slime production allows bacteria to form a
biofilm
• Biofilm is community of bacterial cells that
produce polymeric matrix in huge amount
• May contain one or more species
• Cover non-organic and organic surfaces or living cells
• Form thick layer (up to 1 mm) in which bacteria are
deeply immersed and protected.
• Matrix consists not only of polysaccharides but also
of proteins and nucleic acids
• A common cause of nosocomial infections (HAIs) in
patients with artificial devices
 Some Places Where Biofilms are
Formed

• On the surface of catheters.

• On contact lenses.
• They grow on pacemakers, heart
valve replacements.
• Artificial joints and other surgical
implants.
• Other places.
Capsule, Slime, Biofilm
Stages in Biofilm Formation
 Quorum Sensing

• In biofilms, cell-to-cell signaling through

different molecules exists.

• All cells function together as a one multicellular

organism.

• It is known as “quorum sensing” of bacteria.

• The aim is protection.

 Producers of Biofilms

• Many bacterial species form biofilms.

• Gram-positive and Gram-negative bacteria

use different signals to produce biofilms.

• Fungi can also form biofilms.

 Producers of Biofilms

Staphylococcus aureus biofilm

Pseudomonas aeruginosa
on an indwelling catheter
biofilm
(urinary bladder catheter)
 Producers of Biofilms

Scanning electron micrograph of fungal biofilm on

bandage contact lens (soft contact lens)
 Importance

• Biofilms are related with

nosocomial infections.

• Over 65% of nosocomial (hospital-

acquired) infections are caused by
biofilms.
 Problems in Treatment

• Bacteria growing in a biofilm are

highly resistant to antibiotics.

• The antibiotic can not reach the bacteria.

• Standard antibiotic therapy is often

unsuccessful and the only recourse may be
to remove the contaminated implant.
 Bacterial Spores (Endospores)

• Modified Gram-positive bacterial cells

• In state of anabiosis, dormant cells

• Defensive strategy for experiencing

adverse and harsh conditions

• Problem for medicine and food industry

 Spore Forming Bacteria

• N.B. Only two genera of bacteria can form

spores, both of which are Gram-positive
rods:
– genus Bacillus
– genus Clostridium
 One Cell - One Spore
(Sporulation)

,
 Time

• The sporulation process requires 6 to 8

hours for completion.

• The germination of spores into the

vegetative state takes approximately 90
minutes.

• After the germination process begins, the

spore will take up water, swell, shed its
coats, and one spore produce one new
vegetative cell.
Sporulation
 Spore Structure

Multilayered protective coat of spores

Comparison Between Vegetative Cell and Spore

Shape

Са++ Low High

Unique
Dipicolinic acid No
structure
Water 80-90 % 10-25%
Resistance
Chemicals, temperature, Low High
radiation

Metabolism High level Absent

Lysozyme Susceptible Resistant
 Location of the Spores
• The location of the spore within a • Variations in deformation of
cell is a characteristic and can vegetative cell:
assist in identification of the • Genus Bacillus – do not swell
bacterium. the cell
• Variations in spore location: • Genus Clostridium – make
– terminal (C. tetani) deformation of the cell
– sub-terminal (C. perfringens)
– central (Bacillus anthracis)
Sub-terminal Spore (C. perfringens)
 Methods for Staining of the Spores

Gram stain of Bacillus anthracis

A stained preparation of Bacillus
subtilis - endospores are green forming spores. The bacteria (laid
and the vegetative cell are red end-to-end forming a chain) are
stained violet, but the spores are
unstained.
The Anthrax Spores
 N.B.
Pathogenic organisms sporulate
usually in the environment.
In the soil they last decades.
 Characteristics of the Spores and their
Medical Importance

• Highly resistant to heat; not killed by boiling (100 °C),

but die at 121 °C under pressure
• Medical instruments must be autoclaved (1 atm, 121° C
for at least 15 min)

• Highly resistant to chemicals (most disinfectants).

• Only solutions labeled as sporicidal

• Survive many years in the soil

• Wounds contaminated with soil can be infected
with spores. Development of tetanus or gas
gangrene (C. tetani, C. perfringens) is possible
 Characteristics of the Spores and their
Medical Importance

• No measurable metabolic activity

• Antibiotics are ineffective, there is nothing
to suppress. Moreover, the spores are
impermeable for them.
• Endospores are formed by lack of nutrients
and germinate if they are available.
• Spores are rarely found in the site of the
infection. There are more vegetative forms in
the wounds.
 Storage Granules (Inclusion Bodies)

• Stored nutrients present in excess.

• Polysaccharide granules
• Lipid inclusions
• Metachromatic granules – inorganic
polyphosphates (volutin granules).
• Their presence and amount vary with the
species of bacteria and its metabolic
activity.
 Volutin Granules

Corynebacterium diphtheriae
 Plasmids

• Some bacteria contain plasmids - small

circular double-stranded DNA
molecules.

• They are not essential for cellular survival.

• Plasmid genes often provide a selective

advantages of bacterial cells as the production
of important virulence factors.
Bacterial Chromosome and
Plasmid DNA