Microbiology

References
Murray et al. Medical Microbiology, 2020

Sherris Medical Microbiology 2018
1
Introduction and Classification
of Microorganisms
Prof. Dr. Rıza Durmaz
YİÜ 2022
Introduction
Microbiology is the study of microorganisms, a large and diverse group of
microscopic organisms.
Microorganisms can be subdivided in to four general groups:
– Viruses:
The smallest infectious particles, ranging in diameter from 18 to 600
nanometers,
– Bacteria:
They are prokaryotic organisms
– Fungi
They are eukaryotic organisms
Fungi can exist either in a unicellular form (yeast) or in a
filamentous form (mold)
– Parasites:
All parasites are classified as eukaryotic,
Some are unicellular and others are multicellular.
They range in size from tiny protozoa as small as 1 to 2 µm in
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diameter to tapeworms that can measure up to 10 meters in length 3
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Increasing complexity: viruses →
bacteria → fungi → parasites
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Introduction
Microorganisms have a tremendous impact on all life.
They live in/on our body and our environment including the air, water, and
food
They are responsible for cycling the chemical elements essential for life,
including carbon, nitrogen, sulfur, hydrogen, and oxygen;
– more photosynthesis is carried out by microorganisms than by green plants.
Humans have an intimate relationship with microorganims; more than

90% of the cells in our body are microbes.
– There are nearly 10 times more microbial cells in and on our body
than our own human cells.
– There are a hundred times more microbial genes present in our
microbiome than our own human genes.
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Benefits of microorganisms for humans
Microorganisms are used in the production of fermented

foods and beverages
Microorganisms are generally used industrially for the
production of antibiotics, vaccines and insülin.
Microorganisms in microflora produces vitamin K,
Microbes in the air are responsible for the degradation of
dead cells
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Benefits of bacteria
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Introduction
The estimated total
number of bacteria on the
planet is 1030 or one
nonillion.
Not all microorganims

cause harm
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CLASSIFICATION OF MICROORGANISM
Five-Kingdom System of Biological Classification
Proposed in 1969 by Robert Whitaker
Five kingdoms based on

– the differences of prokaryotic and eukaryotic cells
– the cellular organization (unicellular/ multicellular
– the nutrition ways (photoautotrophic/ kemoheterortophic)
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Five-Kingdom (1969)
1. Kingdom Prokaryotae (Monera):

Oldest known cells.
Lived over 3.5 billion years ago.
Lack a nucleus and membrane bound organelles.
The other four kingdoms are eukaryotes. Have a true nucleus
and membrane bound organelles.
2. Kingdom Protista: Mostly unicellular, lack tissue organization.
3. Kingdom Fungi: May be unicellular (yeasts) or multicellular (molds).
4. Kingdom Plantae: Multicellular, photosynthetic.
5. Kingdom Animalia: Multicellular, heterotrophs that ingest food through
a mouth or oral cavity.
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Five-Kingdom Classification System
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Seven Kingdoms-2015
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Phylogeny:The Three Domain System
by Carl Woese (1990)
Domain is above the kingdom, domain includes kingdom
Phylogeny refers to the study of evolutionary relationship of the organisms
TWO OF THESE THREE DOMAINS INCLUDE PROKARYOTEIC CELLS

– There are two types of prokaryotic cells, based on differences in their ribosomal
RNA, cell walls, and metabolism
1. Eubacteria: “True bacteria”.

• Cell wall contains peptidoglycan that include muramic acid.
• Sensitive to antibiotics.
2. Archaeabacteria: “Ancient bacteria”
• Cell walls lack peptidoglycan, resistant to antibiotics.
• tRNA doesn’t have T(timin)
• Live in extreme environments
•Present in human microbiota
Three kingdoms:
1. Methanogens: Strict anaerobes that produce methane.
2. Extreme halophiles: Require high salt concentrations.
3. Thermoacidophiles: Live in hot, acidic environments.
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3.Eukarya
Animalia:
– Multicellular; no cell walls; chemoheterotrophic
Plantae:
– Multicellular; cellulose cell walls; Usually photoautotrophic
Fungi:
– Chemoheterotrophic;
Unicellular or multicellular; cell walls of chitin;
develop from spores or hyphal fragments
Protista: A catchall kingdom for eukaryotic organisms that do not fit other
kingdoms
– Protozoa: Unicellular nonphotosynthetic protists:
Four groups:
– Flagellates,
– Amebae,
– Ciliates,
– Sporozoa
– Algae,
– Slime molds
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Algae
Formerly, all algae were thought to contain chlorophyll
Modern taxonomic approaches have recognized that some

algae lack chlorophyll and have a free-living heterotrophic or
parasitic life style.
Many algal species are unicellular microorganisms.
Other algae may form extremely large multicellular structures
Protothecosis is a disease of dogs, cats, cattle, and rarely
humans caused by a type of algae, Prototheca, that lacks
chlorophyll
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Slime mold or slime mould is an informal name given to several kinds
of unrelated eukaryotic organisms that can live freely as single cells, but
can also aggregate together to form multicellular reproductive
structures.
Slime molds were formerly classified as fungi but are no longer

considered part of that kingdom.
Although not forming a single monophyletic clade, they are grouped

within the paraphyletic group, kingdom Protista.
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Phylogenetic Relationships of
Prokaryotes
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Differences Between Eukaryotic and
Prokaryotic Cells
Prokaryotes Eukaryotes
Cell size 0.2-2 µm in diameter 10-100 µm in diameter
Nucleus Absent Present
Membranous
Organelles Absent Present
Cell Wall Chemically complex When present, simple
Ribosomes Smaller (70S) Larger (80S)
DNA Single circular Multiple linear
chromosome chromosomes (histones)
Cell Division Binary fission Mitosis
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Difference between Eukaryotic and Prokaryotic cells
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Hierarchy of Taxonomic Categories
DOMAIN
Kingdom
Phylum or Division (Bacteria)
Class
Order
Family
Genus
Species
subtype
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E.coli
Kingdom: Prokaryotae
Division : Gracilucutes
Class : Scotobacteria
Order: Eubacteriales
Family: Enterobacteriaceae
Genus: Escherichia
Species: coli
subtype. Escherichia coli O157:H7
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Nomenclature of microorganisms
nomenclature: Universal system for naming and
classifying living organisms.
Binomial nomenclature: Each organism (species) has

a two part name: genus + specific.
Genus name: Always capitalized, always a noun.
species name: Always lower case, usually an adjective.
Names are either italicized or underlined.
Penicillium notatum
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Scientific Names
Source of Genus Source of
Scientific Binomial Name species name
Klebsiella Honors Edwin Klebs The disease

pneumoniae
Pfiesteria piscicida Honors Lois Pfiester Disease in fish
Salmonella Honors Daniel typh- in mice (muri-)

typhimurium Salmon
Streptococcus Chains of cells Forms pus (pyo-)
pyogenes (strepto-)
Penicillium Tuftlike (penicill-) Produces a yellow
chrysogenum (chryso-) pigment
Corkscrew-like Honors Oswaldo
Trypanosoma cruzi (trypano-, borer; Cruz
soma-, body)
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Classification systems
– Phenotypic classification:
Microscopic and macroscopic morphologies
Biochemical tests
Biotyping
Serotyping
Antibiogram patterns
phagetyping
– Numerical Taxonomy (also called computer taxonomy or
taxometrics).
Use a large number (>100) of taxonomically useful characteristics
The computer clusters different strains at selected levels of overall
similarity (>80% at the species level)
provide persentage of similarities for all strains within each cluster.
– Phylogenetic classification :
understanding of evolutionary relationships among
microorganisms
Comparison of the nucleotid sequence of 16S rRNA
Plasmid analysis
Analysis of Chromosomal DNA fragments
Protein analysis
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Identification
Use of a classification scheme to identify the
causative agent of a disease
Morphological characteristics Useful for identifying eukaryotes
Differential staining Gram staining, acid-fast staining
Biochemical tests Determines presence of bacterial enzymes
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Identifying Bacteria
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Some common Definitions
Serotyping
Many bacteria possess antigens Slide agglutination
that are unique, and antibodies
used to detect these antigens
are powerful tools for their
identification
Useful in determining the identity
of strains and species, as well
as relationships among
organisms.
Example
– Slide agglutination
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Phage Typing
Identification of bacterial
species and strains by
determining their
susceptibility to various
phages.
Bacteriophages are highly
specific for bacteria.
Bacterial culture is mixed
with different phages. The
bacteriophage lyses
unknown bacterium which is
determinative.
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Antibiogram patterns: Patterns of susceptibility to various
antibiotics
Biotyping: The use of biochemical tests to identify clinically

significant isolates.
– This method can also be used for subdividing groups of organisms
beyond the species level
Biovars are strains that are differentiated by biochemical or
other non-serological means.
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An isolate: a pure culture derived from the original
sample.
– Isolate is a general term for a pure culture of bacteria obtained

by subculture of a single colony from a primary isolation plate,
presumed to be derived from a single organism, for which no
information is available aside from its genus and species.
Culture: Grown in laboratory media
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Bacterial species: a collection of strains that share many common
phenotyic characteristics.
– Bacterial species can be defined as a group of bacteria that exhibit ≥
99% 16S rRNA relatedness.
– Bavterial genus can be defined as a group of bacteria that exhibit ≥97%
16S rRNA relatedness
-
Bacterial Strain (A subgroup of a bacterial species ):

A strain is a descriptive subdivision of a species.
Clone: Population of cells derived from a single cell
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References
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Bacterial Cell Structure
Prof.Dr. Rıza Durmaz
YİÜ-2022
1
Objectives
➢ Discuss the basic parts and function of microscopes,
➢ Define the cell wall in prokaryotes
➢ Describe the functions of cell wall
➢ Determine the differences of cell wall between gram

positive and gram negative bacteria and mycobacteria
➢ Describe the functions of cytoplasmic membrane
➢ Define the bacterial cytoplasm and internal structures :

chromosome, plasmid, inclusion bodies, ribosome and endospore
2
MICROSCOPY
➢ Microscopy is used in microbiology for two basic purposes:
⚫ the initial detection of microbes and
⚫ the preliminary or definitive identification of microbes.
➢ The microscopic examination of clinical specimens is used to detect

⚫ bacterial cells, fungal elements, parasites (eggs, larvae, or adult forms), and viral
inclusions present in infected cells.
➢ Characteristic morphologic properties can be used for the preliminary

identification of most bacteria and are used for the definitive identification
of many fungi and parasites.
➢ The microscopic detection of organisms stained with antibodies labeled

with fluorescent dyes or other markers has proved to be very useful for
the specific identification of many organisms.
3
OPTICAL METHODS
➢ The light microscopes
⚫ Bright-field microscope
⚫ Dark-field microscope
⚫ Phase contrast microscope
⚫ Differential interference contrast microscope
➢ Ultravolet rays
⚫ Fleurescent
⚫ Confocal
➢ The Electron microscope
⚫ Transmission electron microscopes,
• electrons such as light pass directly through the specimen,
⚫ Scanning electron microscopes,
• electrons bounce off the surface of the specimen at an angle and a three-
dimensional picture is produced
4
Figure 3.13
Principles of Light Microscopy
➢ They use visible light as a source of illumination
➢ Thier maximum magnification is 2000 times
⚫ Magnification- occurs in two phases
• Objective lens- forms the real image
• Ocular lens- forms the virtual image
• Total power of magnification= the power of the objective X the

power of the ocular
➢ Their resolution pover is 0.2 μm
➢ Three different objective lenses are commonly used:
➢ low power (10-fold magnification): can be used to scan a specimen;
➢ high dry (40-fold): is used to look for large microbes such as parasites and
filamentous fungi; and
➢ oil immersion (100-fold): is used to observe bacteria, yeasts (single-cell stage of
fungi), and the morphologic details of larger organisms and cells.
Resolution
➢ Resolution- the ability to distinguish two adjacent objects or points
from one another
➢ Also known as resolving power

⚫ Resolving power (RP) = Wavelength of light in nm
2 x Numerical aperture of objective lens
⚫ Under ideal conditions, resolving power of the light microscope is

about half of the wavelength of the light being used.
⚫ With yellow light of a wavelength of 0.4 nm, the smallest

separable diameters are about 0.2 μm
⚫ Shorter wavelengths provide a better resolution
⚫ Oil immersion lenses increase the numerical aperture

Bright-Field Microscopy
➢ Most widely used in microbiology
➢ These microscopes generally employ a 100-
power objective lens with a 10-power ocular
lens,
⚫ Magnifying the specimen 1000 times
⚫ Particles 0.2 μm in diameter are
therefore magnified to about 0.2 mm
and so become clearly visible.
➢ The specimen produces an image that is
darker than the surrounding illuminated field
➢ These microscopes can be used with
live, unstained and preserved, stain
specimens
Dark-Field Microscopy
➢ A bright-field microscope can be adapted to a dark-field microscope
by adding a stop to the condenser
⚫ The stop blocks all light from entering the objective lens except for
peripheral light
⚫ only light that is scattered by objects on the slide can reach the eye
➢ The specimen produces an image that is brightly illuminated

against a dark field
➢ It does not allow for visualization of fine internal details of cells
➢ This technique has been particularly useful for observing organisms

such as Treponema pallidum
Phase-Contrast Microscopy
➢ Transforms bright changes in light waves
passing through a specimen into
differences in light intensity
➢ Allows differentiation of internal

components of live, unstained cells
➢ Useful for viewing intracellular structures

such as bacterial spores, granules, and
organelles
➢ This creates a three-dimensional image of
the organism or specimen
Differential Interference
Microscopy
⚫ Uses a differential-interference
contrast (DIC) microscope
⚫ The image is colorful and

three-dimensional
⚫ Allows for detailed view of live,

unstained specimens, such as
spores, vacuoles, granules
Fluorescence Microscopy
➢ Includes a UV radiation source and a filter that protects
the viewer’s eyes
➢ Used with dyes that show fluorescence under UV rays
➢ Forms a colored image against a black field
➢ Used in diagnosing infections caused by specific
bacteria, protozoans, and viruses using fluorescent
antibodies
Confocal Microscopy
➢ Allows for viewing cells at higher
magnifications using a laser beam of
light to scan various depths in the
specimen
➢ Most often used on fluorescently

stained specimens
➢ Unstaining specimens can
be visualized
Electron Microscopy
➢ Magnification can be extremely high (between
5,000X and 1,000,000X for biological specimens)
➢ Allows scientists to view the finest structure of

cells
⚫ Viruses, cell wall, cytoplasm…
➢ Two forms: transmission electron microscope

(TEM) and scanning electron microscope (SEM)
➢ Bacteriacan be distinguished from one
another by their following characteristics:
⚫ morphology (size, shape, staining characteristics)
⚫ metabolic characteristics
⚫ Antigenic characteristics
⚫ Genetic characteristics
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The size of bacterial cell
➢ Perhaps the most obvious structural characteristic of bacteria
is (with some exceptions) their small size.
➢ For example,
⚫ Escherichia coli cells, an "average" sized bacterium, are
about 2 micrometres (μm) long and 0.5 μm in diameter

⚫ Staphylococcus aureus: 1 μm in diameter
⚫ Bacillus anthracis: 1 μm in diameter and 3-4 μm in length
⚫ Brucella abortus: 1.2 μm in length
⚫ The average diameter of the Erythrocytes is ~7 µm

⚫ Granular leukocytes are ~ 12-15 µm in diameter
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Bacterial Shapes ,Arrangement
Bacteria display a large diversity of cell morphologies and arrangements
1 Bacilli
a. Bacillus (rod shape)
b. Streptobacillus (chain formed bacillus e.g.Bacillus subtilis)
c. Coccobacillus (very short and plump shaped bacillus e.g. Brucella)
2. Cocci
a. Coccus (spherical shape)
b. Diplococcus (two cells together e.g. Neisseria meningitidis)
c. Streptococcus (chain formed coccus e.g. Streptococcus pyogenes)
d. Staphylococcus (grape-like e.g. Staphylococcus aureus)
e. Sarcina (packets of coccus)
f. tetrads ( cocci in packets of four . e.g.Micrococcus species)
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3. Spiral – helical, comma,
twisted rod
• vibrio – gently curved or comma
shaped spiral ( Vibrio cholera)
• spirillum – thick, rigid,
spiral; motility with flagella
(Campylobacter, Helicobacter)
• spirochete– snake like
thin, flexible spiral;
motility with axial filament
(Treponema pallidum, Borrelia)
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Arrangement of cells is dependent on pattern
of division and how cells remain attached
after division.
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Stains/dyes
➢ Stains (dyes) are chemicals containing chromophores groups
that impart color
➢ Stains are generally salts in which one of the ions is colored
➢ Based on the charges:
⚫ A basic dye consists of a colored cation (positively
charged) with a colorless anion

• Examples: crystal violet, safranin, basic fuchsin and methylene
blue
⚫ Acid dyes have negatively charged chromophores.
• They stain the background and leave the microbe transparent.
Examples: Sodium eosinate, Nigrosine and congo red.
⚫ Neutral stain/dyes – stain with both charges
Based on function of stain
➢ 1. Simple staining –Staining can be performed with basic
dyes such as crystal violet or methylene blue
⚫ They are useful solely for increasing contrast so that morphology,
size, and arrangement of organisms can be determined
➢ 2. Differential staining - more than one dye is used-
⚫ Differentiation among bacteria is possible-
⚫ Eg. Gram’s staining, Acid-fast staining.
➢ 3. Special staining – more than one dye used -

Special structures are seen.
⚫ Eg. Capsule staining, Spore staining
Gram Stain
➢ Gram staining allows clinicians to
distinguish between two major classes of
bacteria and to initiate therapy
➢ Gram positive
➢ Gram negative
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Gram staining method
➢ Crystal violet: 1 min
➢ Wash with water
➢ Lugol: 1 min
➢ Wash with water
➢ Alcohol: 30 s
➢ Wash with water
➢ Fuchsine: 30 s
➢ Wash with water
➢ Dry
30
Principles of the Gram stain
➢ Gram (+) bacteria turn purple, the stain
gets trapped in peptidoglycan layer
➢ Gram(-) bacteria have a thin peptidoglycan

layer which does not retain crystal violet,
cells counterstained with safranin and
turned red.
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Gram positive cells
32
Gram negative cells
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Bacterial Cell Structures &
Functions
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Bacterial Cell Structure
➢ Appendages - flagella, pili or fimbriae
➢ Surface
layers - capsule, cell wall, cell
membrane
➢ Cytoplasm -nuclear material, ribosome,
mesosome, inclusions etc.
➢ Special structure - endospore
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APPENDAGES
Flagella:
➢ a lash-like appendage
➢ composed of helically coiled protein
subunits(flagellin)
➢ Some rods and spiral form bacteria have
flagella
➢ Bacteria may have one or several their
surfaces
function:
a. Motility: Swimming toward food or away
from poisons -chemical stimuli (chemotaxis)
b.Antigenic: Carry antigenic and strain
determinants
origin : cell membrane, flagella attach to the
cell by hook and basal body
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Localization of flagella
A)Monotrichous bacteria have a single flagellum (e.g., Vibrio cholerae).
B)Lophotrichous bacteria have multiple flagella located at the same spot
on the bacterial surfaces which act in concert to drive the bacteria in a single
direction.
C)Amphitrichous bacteria have a single flagellum on each of two opposite
ends
D)Peritrichous bacteria have flagella projecting in all directions (e.g., E. coli)

.
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one polar flagella,monotrichous both ends, bipolar: Amphitrichous
tuft at one end,lophotrishous all around bacteria,peritrichous
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2. Fimbriae and Pili
➢ Fimbriae: hairlike structures
⚫ They are composed of protein subunits (pilin)
⚫ Shorter, straighter, smaller than flagella
⚫ Not coiled in structure
⚫ Found on many Gram negative (-) bacteria and some Gram-
positive bacteria.
⚫ Several hundred are arranged peritrichously
a) function:
Adherence to other bacteria or to the host (alternative names
adhesins)
⚫ for the adherence of bacteria, specific receptor sites are required on the host
cell membrane.
➢ Not involve in motility.
➢ Prevent phagocytosis
➢ Fimbria is important virulence factor , Some pathogens cause
diseases due to this Antigenic characteristic.
e.g: E. coli, N. gonorrhoeae
b) Origin: Cell membrane
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pili
➢ Sex pili (F pili):
If bacteria have pili, they
are F(+) or donors of F
factor.
➢ It is necessary for
bacterial conjugation, to
transfer DNA from one
cell to another.
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Axial Filaments
➢ They are present in spirochetes ( Treponema pallidum
causes syphilis)
➢ Their function is motility – gliding motility
➢ They look like as bundles of fibres at the ends of the cell
➢ They are structurally similar to flagella
➢ They locate under an outer membrane
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II. CELL SURFACE LAYER
1. Capsule
2. Slime layers or a glycocalyx are similar to capsules
but are more diffuse layers surrounding the cell
➢ Capsule is well organized layer and not easily washed

off
➢ Slime layer, unorganized material, nonuniform and
loosely adherent, that is easily removed.
➢ They give mucoid growth on agar plate
Capsule:
➢ It consists of polysaccharide or protein layers
⚫ Most of them have only polysaccharide.
⚫ B. anthracis has a capsule of poly-D-glutamic acid,
⚫ S. pyogenes has a capsule made of Hyaluronic acid
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Capsule and Slime layer
➢ Function:
⚫ Resistant to host phagocytosis, Major virulans factor.
⚫ (e.g.Streptococcus pneumoniae, Haemophilus influenzae,
Neissseria meningitis).
⚫ Protect against desiccation,
⚫ Attachment to surface of solid objects.
⚫ The capsule is antigenic.
• can be used for vaccine development
• can be used to serotyping of bacteria
⚫ Act as an barrier to toxic hydrophobic molecules (such as
detergents)
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Biofilm formation
(surface bio-materials)
Biofilm is a special bacterial adaptation that facilitates
colonization
➢ Sticky web of polysaccharides in glycocalyx binds the cells together and to

the surface
➢ Biofilm is composed of numerous numbers of microorganisms

➢ Biofilm is produced by certain bacteria (Coagulase negative staphylococci,
S. aureus, P. aeruginosa, S.mutans )
➢ Biofilm forms at interfaces of Plastic surfaces or prosthetic devices
➢ Within the biofilms, bacteria protect themselves from the immune system
and from the effect of antibiotics (antibiotic resistance!)
➢ Biofilms are important in human infections that are persistent and difficult to
treat.
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Bacterial Cell Wall
➢ Most prokaryotes have peptidoglycan

(murein=mucopeptide) layer.
⚫ The exceptions are
• Archaeobacteria (which contain pseudoglycan)

and
• Mycoplasmas ( no cell walls)
Peptidoglycan is unique to prokaryotic cells
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Functions of the Cell Wall
➢ Peptidoglycan provides rigidity,
➢ it determines the shape of bacterial cell.
➢ Contribute to pathogenicity (e.g. LPS in Gram negative
bacteria)
➢ Protection from toxic compounds
➢ Distinguish gram positive bacteria from gram negative
bacteria
➢ Protection for osmotic shock (lysis)
⚫ If water moves in → lysis (in a hypotonic environment)
⚫ If water moves out → plasmolysis (shriveling) (in a
hypertonic environment)
Function of Cell wall-2
➢ The cell wall can be removed experimentally
⚫ Lysozyme – hydrolyzes PTG (peptidoglycan)
⚫ Penicillin – inhibits PTG syntesis
⚫ Result:
• Gram-positive → protoplasts
• Gram-negative → spheroplasts (OM intact and peptidoglycan
has been removed)
⚫ Both forms are osmotically sensitive →
⚫ They must be maintained in an isotonic solution
• L forms: If such cells are able to grow and divide, they are
called L forms
L forms
➢ L form: are difficult to cultivate and require specific agar
medium having the right osmtic strength.
➢ Some L form can revert to the normal bacillary form.
⚫ Others are stable and never revert.
➢ Some bacterial species produce L forms spontaneously.
➢ L forms may produce chronic infection in host
➢ L- form infections are relatively resistant to antibiotic
treatment,
➢ Their revision to the bacillary form can produce relapse

of the infection
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Structure of Cell Wall
➢ murein, mucopeptide or peptidoglycan
(all are synonyms )
➢ It is made up of linear polysaccharide chains cross-linked
by peptides
➢ The backbone of the peptidoglycan consist of N-
acetylglucosamin (NAGA) and N-acetylmuramic acid
(NAMA) connected by  1-4 linkages.
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CELL WALL cont…
➢ TETRAPEPTIDES
➢ Muramic asid (NAM) residues are linked to short
peptides.
⚫ This tetrapeptid is unusual because it contains both
L and D form amino asids.

⚫ D-form a.a. is found in nature only in prokaryotic cell
wall.
⚫ The composition of peptides varies one bacterial
species to another.
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➢ The tetrapeptide side chains of all species have certain
important features;
⚫ Most have L-alanine at position 1(attached to NAM), D-
glutamate at position 2, and D-alanine at position 4

⚫ Poisition 3 is the most variable one
• Most gram-negative bacteria have diaminopimelic acid

at this position
• Gram-positive bacteria usually have L-lysine at this

position, however some may have diaminopimelic acid
or another amino acid.
• Diaminopimelic acid is a unique element of bacterial cell

walls.
• It is never found in the cell walls of Archaea or eukaryotes 56
Gram positive bacterial cell wall
➢ Thick cell wall is consist of multiple peptidoglycan layers (as many
as 40 sheets in gram-positive, only 1 or 2 sheets in gram-negative)
⚫ In gram positive; pentaglycin bridge is used between tetrapeptide
linkages to lengthen the cross-link( interpeptid bridges ).
In Gm (–) cell wall; interbridge is used between tetrapeptide
linkages
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➢ The cross-linking reaction is catalyzed by membrane
bound transpeptidases.
⚫ Related enzymes, D-carboxypeptidases,
➢ The transpeptidases and carboxypeptidases are called

penicillin-binding proteins (PBPs)
⚫ they are targets for penicillin and other β-lactam
antibiotics.
⚫ Penicillin and related β-lactam antibiotics bound to
these enzymes and inhibit cross-linking reaction

➢ Vancomycin binds to the D-Ala-D-Ala structure to block
transpeptidations/transglycolysation
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Action of penicillin on cell wall synthesis
Penicillin and other β-lactam antibiotics act by inhibiting penicillin-binding
proteins, which normally catalyze cross-linking of bacterial cell walls.
This is achieved through binding of penicillin to the enzyme DD-

transpeptidase. As a consequence, DD-transpeptidase cannot catalyze
formation of these cross-links
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The peptidoglycan monomers are synthesized in the cytosol and then attached to a
membrane carrier BACTOPRENOL ( a lipid)
BACTOPRENOL transports peptidogycan monomers across the cell membrane where
they are inserted into existing peptidoglycan.
Schematic representation of peptidoglycan biosynthesis and its inhibition by antibiotics.
Gram positive cell wall
Gram positive bacterial cell wall has teichoic acid
Teichoic acid (TA): Polymer of ribose or glycerol joined by phosphate
groups
➢ There are two types of Teichoic asids:
⚫ Wall teichoic acids are covalently linked to peptidoglycan (WTA)
⚫ Lipoteichoic acids are covalently linked to membrane (LTA
61
Functions of teichoic acid
⚫ LTA are common surface antigens of Gram positive
species
⚫ Responsible for the negative charge of the cell surface
⚫ Participate in the supply of Mg++ to the cell by binding Mg++
⚫ Regulate normal cell division
⚫ Resistance to environmental stresses,
• such as heat , low osmolarity , antimicrobial peptides, antimicrobial
fatty acids, cationic antibiotics, and lytic enzymes produced by the
host, including lysozymes
⚫ LTAs also act as receptors for phage particles
⚫ LTA promote attachment to other bacteria and to specific receptors on
mammalian cell surfaces (adherence). Major virulance factor
⚫ WTA act as a cement to strengthen of peptidoglycan
62
Gram positive bacterial cell wall
➢ The peptidoglycan can be degraded by

treatment with lysozyme (in serum,tissues and
secretions and in the phagocytic lysosome)
➢ Without the peptidoglycan, the bacteria lyse.
➢ Removal of cell wall will produces a protoplast.
➢ Protoplast lyses unless osmotically stabilized.
63
Structure of the Gram positive Cell
Wall
64
Gram negative cell wall
➢ Contains two layers external to the cytoplasmic
membrane
⚫ thin peptidoglycan layer
⚫ Outer membrane
No teichoic or lipoteichoic acids
65
Gram negative bacterial cell wall:
Outer membran
➢ structure: lipopolysaccharide (LPS), phospholipid and
proteins.
➢ the external to the peptidoglycan layer
➢ unique to Gram negative bacteria
➢ maintains the bacterial structure
➢ permeability barrier to large molecules (e.g proteins
such as lysozyme) and hydrophobic molecules
➢ Protects from digestive system of the host
66
Outer membrane
➢ LPS molecules are found in the outer leaflet of outer
membrane
➢ LPS consists of three structural sections:

⚫ Lipid A ,
⚫ core polysaccharide and
⚫ O antigen.
• Lipid A is responsible for the endotoxin activity and it is

essential for bacterial viability.
• Endotoxin is a stimulator of innate and immune
responses
➢ LPS causes fever and can cause shock

67
Outer membrane
➢ The variety of protein found in outer membrane
⚫ Transmembrane proteins (porins) form pores
• Pores allow the diffusion of hydrophilic molecules

(metabolites and small hydrophilic antibiotics) throw the
membrane
⚫ Large or hyrophobic antibiotics and proteins (e.g lysosyme)
cannot pass
• The gram-negative cell wall also has different secration
sytems, including the type I, II, III, IV, V , VI..secretion
devices.
⚫ Structural proteins and reseptor molecules for
bacteriophages
⚫ Lipoproteins: Outer membrane is connected to

peptidoglycan by lipoproteins
68
Outer membran..
➢ Outer membrane can be disrupted by antibiotics and
removal Mg and Ca ions (with EDTA or tetracyline )
➢ Distruption of outer membrane weakens the bacteria and

allows the permeability of large hydrophobic molecules
➢ Disruption of the outer membrane can provide entry of

lysozyme to produce spheroplast
⚫ Sferobast forms are sensitive osmotically.
69
Protoplast/Sferoplast
70
The Gram-negative outer membrane(1)
71
Gram negative bacterial cell wall:
periplasmic space: the area is between the
external surface of the cytoplasmic membrane
and the internal surface of the outer membrane
it contains:
➢ hydrolytic enzymes (for breakdown of large
macromolecules for metabolism)
➢ lytic virulence factors e.g collagenases,
hyaluronidases and - lactamase
➢ Sugar transport systems and binding proteins
facilitate the uptake of different metabolites
72
Comparison of Gram positive and Gram negative cell walls
Characteristic Gram positive Gram negative

Number of major layers One Two
Chemical composition Peptidoglycan Peptidoglycan

Teichoic acid Lipopolysaccharide
Lipoteichoic acid Lipoproteins
Mycolic acids in some cells Porin proteins
Phospholipids
Overall thickness Thicker (20-80 nm) Thinner (8-11nm)
Outer membrane no yes
Periplasmic space no yes
Permeability to molecules More penetrable Less penetrable

73
Atypical Cell Walls
➢ Mycobacteria have a peptidoglycan layer
( slightly different ) surrounded wax-like lipid coat of
mycolic acid, cord factor- glycolipid of trehalose and two mycolic acid-
sulfolipids.
• pathogenicity and high degree of resistance to certain chemicals
and dyes
• basis for acid-fast stain used for diagnosis of infections caused
by these microorganisms
➢ Some have no cell wall i.e. Mycoplasma

⚫ cell membrane is stabilized by sterols
⚫ pleomorphic
74
75
Cell Membrane
➢ It is typical «unite membrane» composed of phospholipids and
different kind of proteins
⚫ Proteins account for approximately 70% of the mass of the membrane
➢ Similar to eukaryotic cell membrane but some differs.
⚫ e.g. sterols such as cholesterol in Eukaryot not in Prokaryot (except:
Mycoplasmas)
76
Cell Membrane
Function: Many functions attributable to organelles in eukaryotes
a. Selective permeability (contains transport proteins)
b. Electron transport, energy production
c. Contain enzymes to synthesis and transport cell wall substances and
for metabolism
d. Secret hydrolytic enzymes
e. Regulate cell division.(a coiled membrane, the mesosome)
mesosome act as an anchor to bind and pull apart doughter
chromosomes during cell division
f. include actin like protein flaments which determine shape of the
bacteria (e.g Treponema)
77
➢ EFFLUX PUMPS are proteinaceous transporters localized in
the cytoplasmic membrane of all kinds of cells.
➢ Efflux pumps are capable of moving a variety of different toxic compounds
out of cells, such as antibiotics, heavy metals, quorum sensing signals,
and bacterial metabolites via active efflux.
78
79
Functions of
the cytoplasmic membrane(1)
80
Bacterial Internal Structures
➢ Cell cytoplasm:
⚫ dense gelatinous solution of sugars, amino acids, and
salts
⚫ 70-80% water
• serves as solvent for materials used in all cell functions
➢ Bacterial chromosome:
⚫ Single,double-stranded circle DNA.
• There are exceptions, (Borrelia burgdorferi and Streptomyces
coelicolor) have a lineer chromosome
⚫ DNA is not in a nucleus but in a discrete area
(nucleoid)
⚫ Absent a nuclear membrane and a mitotic apparatus
⚫ Histones are not present but histone-like proteins exist
in bacteria
⚫ Negatively charged DNA is partially neutralized by
small amines and magnesium ions.
81
The bacterial chromosome and
supercoiling
82
➢ Plasmids
⚫ small circular, double-stranded extrachromosomal
DNA
⚫ free or integrated into the chromosome
⚫ duplicated and passed on to offspring
⚫ Most commonly found Gram (-) bacteria
⚫ not essential to bacterial growth and metabolism
⚫ may encode antibiotic resistance, tolerance to toxic
metals, enzymes and toxins
⚫ used in genetic engineering- readily manipulated and
transferred from cell to cell
83
Ribosomes (70 S)
⚫ made of 60% ribosomal RNA and 40% protein
⚫ consist of two subunits: large (50S) and small (30 S)
⚫ prokaryotic ribosomes differ from eukaryotic’s in size
and number of proteins
⚫ Major targets for antimicrobial drugs.
⚫ site of protein synthesis
⚫ present in all cells
84
Bacteria (70S) Eukaryotes (80S) Mitochondria (55S)
Large subunit 50S 60S 39S
23S 25S 16S
rRNA 5S 5S
5.8S
Proteins 35 45 48
Small subunit 30S 40S 28S
rRNA 16S 18S 12S

Proteins 20 33 29
85
Mesosomes
➢ large invaginations of the plasma membrane, irregular in
shape.
➢ increase in membrane surface, which may be useful as a site

for enzyme activity in respiration and transport.
➢ may participate in cell replication by serving as a place of

attachment for the bacterial chromosome.
86
Inclusions
➢ They appear as refractile bodies in the cytoplasm by a phase

contrast microscope
➢ They are storage of energy or as a reservoir of structural building
blocks
➢ glycogen ( used as carbon source), Elemental Sulfur , Lipid
inclusion, polyhydroxybutyric acid droplets (PHB) are
produced when the sources of N,S, P is limited and there is
excess C in the medium
➢ inorganic polyphosphate is called metachromatic granules or
Volutin granules are characteristic for Corynebacterium). 87
Special Structure: Endospore
➢ Several bacterial genera are capable of forming spores

Spore formers: Bacillus and Clostridium ( Gram + Rods) have
medical importance.
➢ Position: central, sub-terminal and terminal
➢ Spores contain a complete copy of the bacterial DNA,

min.concentration essential proteins and ribosomes, small
water, high calcium dipicolinate
➢ Extremely resistant to heat (due to their dehyrated state and

presence of calcium dipicolinate), UV, most enzymes and
chemicals etc. Decontamination with standard disinfectants of
spores is difficult. 88
ENDOSPORES
sporulation
➢ Depletion of specific nutrients from
the growth medium leads to the
production of a spore.
➢ After duplication of the chromosome,

one copy of the DNA and cytoplasmic
contents (core) are surrounded by the
cortex (a thin peptidoglycan )
➢ Cortex is surrounded by keratin-like

protein coat
➢ Sporulation process requires 6-8

hours .
90
Germination of the spores
➢ Germination is the production of one vegetative
cell from one spore
➢ Vegetative state is stimulated by distruption of
the outer coat by mechanical stress, pH, heat
and requires water and nutrient.
➢ Germination process takes 90 min.
➢ Spore takes up water swell,shed its coats
➢ Produce one new vegetative cell identical to the
original cell
91
DIFFERENCES BETWEEN ENDOSPORES AND VEGETATIVE CELLS
Property Vegetative cells Endospores
Surface coats Typical Gram-positive murein Thick spore coat, cortex, and
cell wall polymer peptidoglycan core wall
Microscopic appearance Nonrefractile Refractile

Calcium dipicolinic acid Absent Present in core
Cytoplasmic water activity High Very low
Enzymatic activity Present Absent
Macromolecular synthesis Present Absent
Heat resistance Low High
Resistance to chemicals and acids Low High
Radiation resistance Low High

Sensitivity to lysozyme Sensitive Resistant
Sensitivity to dyes and staining Sensitive Resistant

93
References
➢ Murray et al. Medical Microbiology, 2020
94
Chlamydia
Prof.Dr.Rıza Durmaz
YBÜ-2022
Chlamydiaceae
 They are small ranging in size from about 0.2 µm to l µm.
 They can pass 0.45 micrometer filters
 They are obligate intracellular parasites
– they were considered viruses
 They have the following properties of bacteria

– Possess inner and outer membranes similar to Gram (-) bacteria
– Contain both DNA and RNA
– They have prokaryotic ribosomes
– They synthesize their own proteins, nucleic acids and lipids
– They susceptible to numerous antibacterial antibiotics.
– They lack a typical bacterial peptidoglycan layer in cell Wall
 Although there is no detectable peptidoglycan in chlamydial cells, genomic
studies have demonstrated an almost complete set of genes for peptidoglycan
synthesis .
22.02.21 2
Cell Wall of Chlamydia (RBs)
22.02.21 3
Physiology and structure
 The members of this group share:
– a unique development cycle,
– a common morphology and
– a common genus-specific antigens, species-specific antigens, and serotype-specific antigen. .
 They have genus-specific lipopolysaccaride (LPS) antigen in their cell wall.
 Their major outer membrane proteins (MOMP) are species- and strain-
specific
 They are energy parasites, using host ATP
 They grow in cultures of a variety of eukaryotic cells lines

– McCoy cells, HL, or Hep-2 cells.
– All types of chlamydiae proliferate in embryonated eggs.
22.02.21 4
Classification of the family Chlamydiaceae
The family Chlamydiaceae consists of two medically important
genera
1)Genus: Chlamydia  2) Genus: chlamydophila

– Chlamydia trachomatis – C.pneumoniae
– C.psittaci
22.02.21 5
Characteristics of these species
C.trachomatis C. pneumoniae C. psittaci
Inclusion morphology Round, vacuolar Round, dense Large, variable

shape,dense
Glycogen in inclusions Yes No No
Elementery body morphology Round Pear-shaped, Round

round
Susceptibile to sulfonamides Yes No No
Plasmid Yes No Yes
Serovars 15 1 ≥4
Natural host Human Human Birds
Mode of transmission Person to person Airborne person to Airborne bird
mother to infant person excreta to humans
22.02.21 6
 All Chlamydiae have a common reproductive
cycle, forming
 Elementary body;
– Infectious form,
– metabolically inert
– Extracellular spore-like state
 Reticulate body;
– Non-infectious form,
– metabolically active
– obligate intracellular form in eukaryotic cells
 48-72 hour cycle
22.02.21 7
Growth cycle
 The growth cycle initiate when the small (300-
400nm) EBs attached to the microvilli of
susceptible cells,
 After internalization, chlamydia remain in

cytoplasmic phagosomes,
 In 6-8 hours, the EBs reorganise into the larger

(800-1000 nm) metabolically active RBs
 RBs replicate by binary fission (continuous 18-24

h)
 18-24 h after infection, RBs begin reorganizing
into the smaller EBs
 Between 48-72h, host cell ruptures and then

release
22.02.21 the infective EBs. 8
Growth cycle-2
 Histologic stains can detect the
phagosome with accumulated
RBs, called inclusion .
22.02.21 9
Chlamydia
 They may colonize and infect tissues of the eye and
urogenital tract in humans.

 Chlamydia trachomatis causes several important
diseases in humans:
– lymphogranuloma venereum sexually transmitted
disease
– trachoma, a leading cause of blindness worldwide
 Chlamydophila pneumoniae
– a cause of pneumonia
– has been recently linked to atherosclerosis.
22.02.21 10
Rickettsia
22.02.21 11
Rickettsiaceae Family
 Includes the genera:

 Rickettsia and
 Orientia (Orientia tsutsugamushi)
 Obligate intracellular Gram negative bacteria

Rickettsia and Orientia
 They are small,( 0.3 x 1-2 µm)
 Stained poorly with Gram stain; best with
Giemsa or Gimenez
 They grow only in eukaryotic cells
(intracellular parasites)
22.02.21 13
Characteristics
1. Cell wall structures of Rickettsia are similar to Gram (-) rods
• Peptidoglycan layer is minimal
• LPS has weak endotoxin activity.
• Rickettcia is surrounded with loosely adherent slime layer
2. Orientia lacks both peptidoglycan layer and LPS
4. They contain DNA, RNA and enzymes for Krep’s cycle and ribosomes
for protein synthesis
5. Multiplication by binary fission
6. They are inhibited by antibiotics (e.g.tetracyline, chloramphenicole)
22.02.21 14
7.Pathogenic species Rickettsia and
Orientia are maintained in animal and
arthropod reservoirs
8. They are transmitted by arthropod

vectors (e.g.ticks,mites, lice and fleas)
9. Humans are accidental hosts
10.They produce diseases such as typhus

fever, Rocky Mountain Spotted Fever,
22.02.21 15
Growth cycle
 The Rickettsiae enter eukaryotic cells by attaching to host cell surface
receptors and stimulating phagocytosis.
 After engulfment, they degrade the phagosome membrane by producing
a phospholipase and must be released into the cytoplasm, or the
organism will not survive.
 Multiplication in the host cell by binary fission is slow (generation time, 9
to 12 hours).
– Orientia and the spotted fever group of Rickettsia grow in the cytoplasm and nucleus
of infected cells and are continually released from cells by filopod formation
– In contrast, the typhus group accumulates in the cell cytoplasm until the cell
membranes lyse, bacterial release.
 Once these bacteria are released from the host cell, they are
unstable and die quickly.
22.02.21 16
Replication cycle of Rickettsia and
Orientia (Orientia tsutsugamushi)
Rickettsia rickettsii
Rickettsia rickettsii
Ehrlichia, and Anaplasma
– Anaplasma and Ehrlichia spp. are tick-borne pathogens of

the Anaplasmataceae family
22.02.21 20
Characteristics of Ehrlichia, Anaplasma
 They are intracellular bacteria
 They are pleomorphic intravacuolar organisms that replicate in
granulocytes, monocytes, erythrocytes, and platelets.
 Ehrlichia chaffeensis is responsible for human monocytic

ehrlichiosis
 E. ewingii is responsible for human granulocytic ehrlichiosis
 A. phagocytophilum is responsible for human granulocytic
anaplasmosis
 The cell wall structure of Ehrlichia and Anaplasma is

similar to that of Gram (-) bacteria, but peptidoglycan and
LPS are not present.
22.02.21 21
Growth cycle
 After entry into the host cell, they remain in the phagocytic vacuole
– Fusion with lysosomes is prevented
 The bacteria can multiple by binary fission in the phagosome without

exposure to the hydrolytic lysosome enzymes.
 Two morphologic forms of the bacteria exist:
– Small (0.2 to 0.4 µm) elementary bodies and
– Larger (0.8 to 1.5 µm) reticulate bodies.
– A few days after the cell is infected, the replicating elementary bodies
assemble into membrane-enclosed masses called morulae (inclusions).
– Detection of morulae when the cells are stained with Giemsa or Wright stains is a
rapid, specific diagnostic test
Multiple
morulae of
Ehrlichia canis
in DH82 tissue
culture cells
22.02.21 22
Coxiella
 Coxiella burnetii is classified in Coxiellaceae
 It has gram-negative cell wall,
 It grows intracellularly in eukaryotic cells
 Two structural forms of C. burnetii are recognized:
– small cell variants (spore like form) that are resistant to
environmental stress (e.g., heat, desiccation, chemical agents) and
– large cell variants that are the metabolically active form.
 Human infections occur after the inhalation of airborne
particles from a contaminated environmental source or, less
commonly, after ingestion of contaminated unpasteurized
milk or other dairy products.
C. burnetii has been grown in a cell-free environment
22.02.21 23
Mycoplasma and Ureaplasma
 Acholeplasma, Mycoplasma and Ureaplasma
are unique in that they lack a cell wall and
contain sterols in their plasma membranes.
 Three human pathogens
– Mycoplasma pneumoniae
– M. hominis
– Ureaplasma urealyticum
 The smallest free-living bacteria (0.1-0.3 µm).
 They dont have a cell wall,
 Their cell membrane contains sterols.
 Mycoplasma resistant to penicillins, cephalosporins,
vancomycin and other antibiotics (the cell wall inhibitors)
 They may be free-living in soil and sewage,
 They may be inhabitants of the mouth and urinary tract
of humans, or pathogens.
 In humans, Mycoplasma pneumoniae causes primary
atypical pneumonia, also called walking
pneumonia.
22.02.21 25
 Mycoplasmas form pleomorphic filaments
 They can pass through the 0.45-µm filters used to
remove bacteria from solutions
 Organisms divide by binary fission
 They grow on artificial cell-free media
 They contain both RNA an DNA
 They are facultative anaerobic (except M.pneumoniae
which is strict aerobes)
 They require exogenous sterols supplied by animal
serum added to the growth medium.
 They grow slowly, gen.time 1-6 hours
22.02.21 26
 They form small colonies that have a fried –egg appearance
 M.pneumoniae is an exception, its colonies have been described as

mulberry shaped
 Colonies of Ureoplasma are extremely small measuring 10-50µm
 Major antigenic determinants are membrane glycolipids and

proteins.
 These antigens cross-react with human tissues and other
bacteria.
22.02.21 27
22.02.21
22.02.21 29
References
31
Bacterial Metabolism
Prof.Dr. Rıza Durmaz
YBÜ-2022
– Metabolism : The sum of biochemical
reactions required for energy generation and the
use of energy to synthesise cell material from
small molecules
– Metabolism has an energy- generating

component, called catabolism
– Metabolism has an energy-

consuming, biosynthetic component,
called anabolism
22.02.21 2
Overview of cell metabolism
22.02.21 3
Breakdown
Proteins to Amino Acids, Starch to Glucose
Synthesis
Amino Acids to Proteins, Glucose to Starch
22.02.21 4
 Many of the principles of metabolism are universal
 Differences between bacteria and human eukaryotic cells
– Speed.
 Bacteria metabolize at a rate 10 to 100 times
faster.
– Versatility.
 Bacteria use more varied compounds as energy
sources
– Simplicity.
 Bacteria synthesize macromolecules in a
streamlined way.
– Uniqueness.
 Some biosynthetic processes, such as those
producing peptidoglycan, lipopolysaccharide, and
toxins, are unique to bacteria.
22.02.21 5
Bacterial metabolic processes includes several
reactions.
22.02.21 6
Enrty reaction is the capture of nutrients from the
environment. Then, transfer of nutrients from
outsite of the cell to inside
 Fueling Reactions provide the cell with energy and

with precursor metabolites used in bio-synthetic
reactions.
22.02.21 7
Transport of nutrients
 There are three ways
A) Nutrients enter the cell by simple diffusion:
Molecules move across a semipermeable membrane, without the
protein channels helping
Bacteria use simple diffusion to deliver water, oxygen, carbon dioxide and
small nutrients to the cytoplasm.
B) Some transport occurs by facilitated diffusion (FD):

a protein carrier in the cell membrane, participates in
the shuttling of molecules
no energy is involved,
this process can work only with a concentration

gradient of the given solute.
 C) Active transport involves binding proteins and ATP

22.02.21 8
Facilitated diffusion
 A. The membrane carrier can

change conformation after
binding an external molecule
and subsequently release the
molecule to the cell interior
(B).
 There is no energy input,
 Molecules continue to enter

only as long as their
concentration is greater on
the outside.
22.02.21 9
22.02.21 10
Facilitated diffusion
 Two categories of proteins exist that help this type of
diffusion.
 A) Carrier proteins,
– They can be thought of like a taxi cab in a cell membrane
– they shuffle the molecules from one side of the membrane to the
other side.
 B) Channel proteins,
– They resemble tunnels and create a hole across the cell
membrane.
– In these cases, channels open which allow the molecules to flow
through them.
22.02.21 11
Active transport
 Active transport mechanisms involve specific

protein molecules as carriers of particular
solutes,
 The process is energy linked
 Bacteria have multiple systems of active
transport
– Some of which involve ATP-dependent binding
proteins
– Others that require proton pumps driven by
electron transport within the energized cell
membrane.
22.02.21 12
Active transport.
1.The solute-binding
protein binds the
substrate to be
transported and closes to
the transporter complex.
2.The solute binding

which is moved across
the membrane with the
aid of ATP hydrolysis
22.02.21 13
Active transport
 The transport of iron is of particular importance
in virulence.
– There is little free Fe3+ in human blood or other body
fluids, because it is sequestered by iron-binding proteins
(eg, transferrin in blood and lactoferrin in secretions).
 Bacteria must have iron to grow, and their
colonization of the human host requires capture
of iron.
 Bacteria secrete siderophores (iron-specific
chelators) to trap Fe3+; the iron-containing
chelator is then transported into the bacterium by
specific active transport.
22.02.21 14
Major nutritional types of
prokaryotes
22.02.21 15
Heterotrophic types of metabolism
 Animals and many bacteria are heterotrophs,
particularly those that live in the association
with animals.
 Heterotrophy (i.e. Chemoheterotrophy)

– The use of an organic compound as a source of
carbon and energy. (catabolism and anabolism)
22.02.21 16
Carbohydrate Catabolism
 Organisms catabolize carbohydrates as the primary energy source
– Aerobic cellular respiration → Results in complete breakdown

of glucose to carbon dioxide, water and a lot of
ATP
ATP
– Anaerobic respiration & Fermentation → Only partially

breakdown of glucose, into pyruvic acid and organic end products
and a little
ATP
ATP
22.02.21 17
 Fermentation and respiration pathways each
regenerate ATP and NAD+
 Fermentation is the transfer of electrons and

protons via NAD+ directly to an organic
acceptor.
– Pyruvate occupies a pivotal role in fermentation
 Respiration uses electron chain for which

oxygen is usually the terminal acceptor
22.02.21 18
Fermentation
 In fermentation, energy is derived from the
partial oxidation of an organic compound
– Organic intermediates are used as electron
donors and electron acceptors.
– No outside electron acceptors are involved;
– No membrane or electron transport system is

required;
 All ATP is produced by substrate level-

phosphorylation.
22.02.21 19
In the bacteria there are three major
pathways of glycolysis (the dissimilation
of sugars):
 The classic Embden-Meyerhof pathway (EMP),
– Net production is 2 ATP molecules in the
glycolytic conversion of glucose to two pyruvic
acid.
 The phosphoketolase or heterolactic pathway
used by lactic acid bacteria
– Glucose lactic acid+ethanol+CO2+ 1ATP
 The Entner-Doudoroff pathway used by vibrios

and pseudomonads.
– Glucose 2 ethanol+2CO2+1 ATP
22.02.21 20
Embden-Meyerhof fermentations
are distinguished by their end products
a. Homolactic acid fermentation
Pyruvic acid (P.A) -----> Lactic Acid (eg. Streptococci,
Lactobacilli)
b. Alcoholic fermentation
P.A -----> Ethyl alcohol (eg. Yeasts)
c.Mixed acid fermentation (eg. Enterobacteriaceae)
P.A ----->lactic acid
acetic acid
formic acid
succinic acid
ethyl alcohol with gas formation (H2 + CO2)
d. Butanediol fermentation
P.A -----> 2,3, butylene glycol+acetoin (eg. Klebsiella and
Enterobacter, Pseudomonas)
e. Butyric acid fermentations ( clostridia)

f. Propionic acid fermentation
P.A -----> 2 propionic acid, acetic acid, CO2 (eg. corynebacteria,
Propionibacterium and Bifidobacterium)
22.02.21 21
.
End products of fermentation pathways
22.02.21 22
Respiration
 Result in the complete oxidation of the substrate by an
outside electron acceptor.
 Most efficient way to extract energy from glucose

– Net production is 38 ATP
 In addition to a pathway of glycolysis, four structural or

metabolic components are needed for respiration:
1. The tricarboxylic acid (TCA) cycle (the citric acid cycle or

the Kreb's cycle)
1. A membrane and an associated electron transport

system (ETS).
1. An outside electron acceptor : electron acceptor is O2
1. A transmembranous ATPase enzyme (ATP synthetase).

22.02.21 23
Aerobic glucose metabolism
22.02.21 24
Using oxygen in metabolism
creates toxic waste.
Microbes that are able to use aerobic
respiration produce enzymes to detoxify
oxygen:
Catalase: H2O2 ------- H20 and 02

Superoxide dismutase (SOD): oxygen radical ----- H20
and O2
Microbes that don’t make these enzymes

cannot exist in the presence of oxygen.
22.02.21 25
Anaerobic respiration
– Some bacteria can use compounds other than O2 as a
final electron acceptor in electron transfer system :
never be O2
– In the anaerobic respiration, the final electron
acceptors may be SO4 or S or NO3 or NO2 or certain
other inorganic compounds, or even an organic
compound, such as fumarate.
 More efficient than fermentation which uses organic molecules

as an electron acceptors
 It is less efficient than aerobic respiration because it produces

less ATP.
22.02.21 26
22.02.21 27
References
28
Bacterial Growth and
Cultivation
Prof. Dr. Rıza Durmaz
YBÜ-2022
1
Objections
 Define the microbial growth- increase in the size
or number?
 Define the binary fission
 Describe the bacterial growth curve
 Define the generation time, colony, culture and
inoculum
 Determine the culture media and their
differences
 Describe the environmental factors influencing
bacterial growth
2
Microbial Growth
 Refers to increase in the number of microbes
(reproduction) rather than an increase in size of
the microbe.
 Colony= aggregation of cells arising from

single parent cell.
 A colony is a population of cells arising from a
single cell or spore
 Generation/doubling time.The time required

for growth and reproduction of the bacterial
cell
 Interval of time between two cell divisions
 The time required for a bacterium to give rise to 2
daughter cells under optimum conditions
 The time required for one cell to divide into two
cells
3
Bacterial growth
 It is an increase in all the cell components, which ends in
multiplication of cell leading to an increase in population.
 It involves - an increase in the size of the cell & an

increase in the number of individual cells.
 Bacteria divide by binary fission.
4
Generation Time Under Optimal Conditions
(at 37oC)
Organism Generation
Time
Bacillus cereus 28 min
Escherichia coli 12.5 min
Staphylococcus aureus (causes many types of infections) 27-30 min
Mycobacterium tuberculosis (agent of Tuberculosis) 18 – 24 hrs
Treponema pallidum (agent of Syphilis) 30 hrs
5
Bacteria divide by binary fission
6
Logarithmic increase in Cell Count From Binary
Fission
Generation Cell
Number Count
0 1
1 2
2 4
3 8
4 16
5 32
10 1,024
20 1,048,576
7
Standard bacterial growth curve
8
Bacterial growth phases-1
Lag phase:
 When bacteria are added to a medium,
they require time to adapt to the new
environment
 It is a period of intense metabolic activity
especially enzyme synthesis.
 Cells may double or triple in size in preparation for

division.
 Length of Lag phase depends on species, age of

culture and nutrients.
9
Exponential (logarithmic or log) phase
• Cells are most active metabolically and most sensitive to

adverse conditions e.g antibiotics.
•
• a straight line is obtained by plotting the log of the number
of cells in the culture against time.
• this stage is most often used

• for physiological studies,
• for production of a particular enzyme or other
product in industry .
10
Stationary phase
• metabolism slows down, and the rate of growth equals the rate
of death.
• Stationary phase is caused by;

• exhaustion of nutrients,
• accumulation of toxic waste products
• changes in pH or temperature.
• In spore-forming species, spores are made at this stage.
• In other species, granular inclusions are observed and abnormal

forms may appear.
11
Death phase
• Reproduction has usually stopped
• The death rate is certainly greater than any small

amount of growth.
•
• More abnormal cells appear
• Death cells may or may not lyse.
This stage may take weeks or months depending on

species.
12
Cultivation of Bacteria
Culture: Bacteria growing in broth/on

solid medium
13
PURPOSE OF CULTURE
 To isolate bacteria in pure culture.
 Identify those microorganisms:
 Examine the morphological features
 Observe the microscopic properties
 Obtain sufficient growth for preparation of

antigens and for other tests.
 Type isolates by methods such as bacteriophage
and bacteriocin susceptibility.
 Determine sensitivity to antibiotics.
 Estimate viable counts
 Maintain stock cultures. 14
Culture Media
 Culture Medium: any nutrient medium that is designed to support the
growth or maintenance of a microorganisms or cells.
The basic ingredients of media include
carbon source
nitrogen source
inorganic salt
growth factor and
distilled water etc.
 Inoculum: Introduction of microbes into

medium
 Following the bacterial growth, medium is
thereafter referred as “bacterial culture”.
15
Environmental Factors Influencing
Microbial Growth
 Nutritions
 Oxygen
 Temperature
 pH
 Ionic strength & Osmotic Pressure
16
Microbial Nutrition
 Organisms use a variety of nutrients for:
 their energy needs
 to build organic molecules & cellular structures.
 Most common nutrients contain necessary

elements:
 Carbon
 Oxygen
 Nitrogen
 Hydrogen
 These 4 elements make up 95% of dry weight

of bacterium. These are major components of
organic compounds
 The other 5% is composed of Sulphur, Calcium,
Iron, Magnesium, Natrium, Phosphorus .
 Other elements that are needed are minor
essential (trace) elements. Zn, Mn, Mo, Se,
Co, Cu, Ni..
 These elements are needed in extremely small amounts,
can be obtained through water intake. 17
Bacteria are classified on the basis
of oxygen requırements
 Obligate aerobes require oxygen for energy production,
 obligate anaerobes cannot tolerate oxygen due to its toxicity and do not
utilize it for energy production.
 Toxic forms of oxygen are highly reactive and damage DNA and proteins if not
neutralized.
 Facultative anaerobes can maintain life with or without oxygen,

 However, their metabolic efficiency is often reduced in the absence of oxygen.
 Aerotolerant anaerobes prefer anaerobic conditions, but can tolerate

oxygen because they have some form of the enzymes that detoxify
oxygen's poisonous forms.
 Microaerophiles require low levels of oxygen.
18
19
Microbes are described in terms of
their temperature requirements
 Psychrophiles require temperatures below

20°C.
 Mesophiles grow best at temperatures ranging
between about 20°C and 40°C with optimum
around 37°C .
 Thermophiles require temperatures above
45°C with optimum around 55 oC
 Hyperthermophiles require temperatures
above 80°C.
20
21
The effect of temperature on the growth
of mesophile
22
Hydrogen Ion Concentration (pH)
 Organisms are sensitive to changes in pH because
the folding of the proteins and DNA in the cell can
be disturbed.
 the cytoplasm of an organism must maintain a
narrow pH range (6.5 to 7.5) to sustain life
(neutrophiles).
 Some bacteria and many fungi are able to withstand
acidic pH and are considered acidophiles.
 Alkaliphiles are a class of extremophilic microbes

capable of survival in alkaline (pH roughly 8.5-11)
environments, growing optimally around a pH of 10
23
Ionic strength & osmotic pressure
 Osmotic pressure restricts cells to certain environments.
 Osmotic pressure and salt concentration have to be

controlled.
 In a hypertonic environment, osmosis can cause cells to
die .
 obligate halophiles, require high salt concentrations in
the environment to survive.
 These organisms often exists in salt water.
 Facultative halophiles do not require but can tolerate
salty conditions.
24
25
Classification of culture
media
 A variety of media are available for microbiological cultures.
According to function, in general, media can be

classified five types:
 Nutrient agar or broth ( General purpose media)

 Enrichment nonselective media
 Selective media
 Differential media
 Specialized media

26
TYPES OF MEDIA
 Nutrient agar/broth General purpose media will

support the growth of many microorganisms.
27
(Enrichment)
28
Enrichment Media
• Encourages growth of desired microbe by providing
special growth conditions or added growth factors
Thioglycollate
Blood agar
Lysed red blood cells provide Glucose Salts Agar (enriches

unique nutrients in for microbes that can growth
blood/chocolate agar only on glucose and some
inorganic nutrients
Selective Media
• Goal: To chemically (or
physically) suppress MSA
unwanted microbes and

encourage desired
microbes.
Mannitol salt agar : selective for halophiles
with 7% salt (osmotic challenge) and
differential for mannitol fermenters: good for EMB
skin bacterial cultures.
EMB Agar: kills gram positives with eosin and

methylene blue, selective for gram negatives.
Differential for lactose fermenters. Good for MA
growing enterics.
McConkey Agar: supresses gram positives with

crystal violet and bile salts; also differential for
lactose fermenters
Figure 6.9b, c
31
Reducing media/Anaerobic media
 Provide conditions to culturing anaerobes.
 They contain compounds that chemically combine with free
oxygen and remove it from the medium.
 Cooked Meat (RCM) Broth Cooked meat particles used as

a reducing agent which absorbs oxygen
 THIOGLYCOLLATE BROTH Thioglycollate used as a

reducing agent which absorbs oxygen
 Also, the use of anaerobic chambers allows for the

manipulatation of anaerobic bacteria in an oxygen free
environment.
32
Classification of media according to
their AGAR content
 Media used in microbiology labs could be divided into

four groups according to their agar content
 Agar is a biological substance extracted from sea algae

 Agar solidifies the media
 > 90oC it is in liquid phase
 < 40oC it is in solid phase
 No nutritive value
 Not affected by the growth of the bacteria
33
• 1)Liquid media; do not include agar
• 2)Semi-liquid media; include 0,05-0,2 % agar
• 3)Semi-solid media; include 0,3-0,5 % agar
• 4)Solid media; include 1,5-2 % agar
4
3
1 2
Pure Cultures Used To Study Characteristics Of A Particular Species
• A pure culture contains only one species or strain

• A colony is a population of cells arising from a single
cell (CFU) or spore
• A colony-forming unit (CFU) is a single bacterium can
grow and become a colony
How could we observe bacterial
growth?
on agar plate
in liquid medium
in semi-solid medium
36
Bacterial growth in solid
culture could be observed as
colony formation
Size
Shape
Hemolysis
Margin (Smooth, Rough,
Mucoid colony)
Surface
Transparency
Pigment
37
Bacterial Colony Morphologies
 Colonies are divided into three groups

according to their surface configuration;
Smooth (S) colonies

Rough (R) colonies
Mucoid (M) colonies
Smooth colonies
Smooth edged,
circular and
swollen
colonies
Rough colonies
Rough edged and knobby colonies with

wrinkle surface
Mucoid colonies
Generally encapsulated bacteria

produce mucoid colonies
on blood agar plate
observe hemolysis (complete and incomplete)
phenomena around colony.
42
In liquid culture bacterial
growth is observed as turbidity..
Uninoculated Inoculated
Estimating Bacterial Numbers
• Direct Measures
• Plate counts of viable bacterial forming colonies
• Counting low viable bacterial numbers by filtration
• Counting viable bacteria with Most Probable
Number
• Counting bacteria per ml in direct microscopy
• Indirect Measures
• Turbidity/Absorbance with a spectrophotometer
• Metabolic activity tracking conversion of colored
molecules
• Dry weight by weighing a set volume and knowing
weight of one cell
3.in semi-solid medium
It is used to test motility
nonmotile organism
motile organism
Motility
Negative Motility Positive
45
References
46
BACTERIAL GENETICS
Prof Dr Rıza DURMAZ

YBU-2022
The bacterial genome is a total collection of gene
carried both on chromosome and on extrachromosomal
genetic elements (Plasmid)
Bacterial pathogens contain between 600 and 6000 genes
.
– Genes are sequences of nucleotides that have a biologic function.
– Each genome contains many operons, which are made up of genes.
– An operon consists of regulatory elements and protein-encoding
genes
– The regulator gene encodes a repressor protein that binds to the
operator site.
– The binding of the repressor to the operator prevents
transcription of the structural genes.
All bacterial cells are haploid
• Alteration of a gene (mutation) will have a more obvious
effect on the cell.
• The structure of the bacterial chromosome is

maintained by polyamins, rather than by histones.
DNA form
• The total length of E.coli chromosome is about 1 mm.
• The Bacterium itself is only several micrometers in lenghts (1-3 µm).
• The DNA is about 1000 times longer than bacterium and must be
condenseded (supercoiling)
• The double-helical DNA chain is twisted into supercoils and attached to the
cell membrane and/or some central structure at a large number of points.
a) The DNA double helix of b)The circular DNA strands,

most prokaryotes has the already coiled in a double helix,
shape of closed circle are twested second time to
produce supercoils
Bacterial Genetics
➢ DNA floats freely within

cytoplasm.
➢ Prokaryotic DNA is packaged

(coiled) differently than
eukaryotic DNA.
➢ That is why some antibiotics

can target prokaryotic nucleic
acid while not hurting the
DNA of our cells (selective
toxicity).
➢ Quinolones,
➢ Q: Prokaryotic DNA is found

in what two structures?
Structure and Function of
Genetic Material
➢DNA & RNA
➢DNA=deoxyribonucleic acid
➢RNA=ribonucleic acid
➢Basic building blocks:
➢Nucleotides
➢Phosphate group
➢Pentose sugar
➢Nitrogenous base
Structure of DNA
• Double stranded (double

helix)
• Chains of nucleotides
• 5’ to 3’ (strands are anti-
parallel)
• Complimentary base pairing
– A-T
– G-C
DNA Replication
• New DNA synthesis occurs at replication forks and proceeds
bidirectionally.
• New DNA is synthesized semiconservatively, using both

strands of the parental DNA as templates.
• Each copy contains one new and one old strand.

DNA replication
• The replication process requires many enzymes,
– helicase unwinds the dsDNA at the origin,
– DNA primase synthesizes primers to start the process,
– DNA-dependent DNA polymerases, DNA polymerase III synthesize
a copy of the new DNA,
– DNA polymerase I fills the gaps between Okazaki fragments
– DNA Ligase: joins discontinous fragments of lagging strant
– RNAase H: cuts RNA primers
• Topoisomerases (e.g., DNA gyrase) supercoil the DNA.
• Topoisomerases are essential to the bacteria
• Topoisomerases are targets for the quinolone antibiotics.

DNA Replication
• DNA helicase-unzips parental DNA strand that is used as a template
• One strand (leading strand) is copied continuously in the 5’ to 3 ‘ direction,

– DNA polymerase III can add nucleotides only to the 3’ end of RNA primer
• Other strand (lagging strand) must be extended in the 5’ to 3 ‘ direction and

must be synthesized as many pieces of DNA (Okazaki fragments -) using
RNA primers
• The lagging strand DNA pieces are ligated together by the enzyme DNA
ligase
Replication Fork
Bacterial DNA replication
New DNA synthesis occurs at

replication (growing) forks and
proceeds bidirectionally.
DNA synthesis progresses in the 5′ to

3′ direction continuously (leading
strand) or in pieces (lagging
strand).
Assuming it takes 40 minutes to

complete one round of replication
DNA replication in bacteria.
Replication begins at the

origin of replication.
Two replication forks proceed in
opposite directions until they
meet at the replication
termination site (ter)., Ex: E.
coli
Bacterial DNA Replication
• DNA replication begins at a single, defined DNA sequence of 245
bp called oriC
– A protein called DnaA, as a complex with ATP, controls onset of initiation

by binding to specific 9 bp repeats at oriC
– The binding distorts the DNA, leading to the opening of adjacent 13-bp
repeats in the DNA----production of replication bubble
DNA replication..
DNA helicase unwind DNA helixe at each of the two

replication forks
PCR: Polymerase Chain Reaction-In Vitro DNA amplification
Transcription
• Transcribe the information carried in the genetic memory of the
DNA into a messenger RNA (mRNA)
• RNA synthesis is performed by a DNA-dependent RNA

polymerase.
▪ Bacterial RNA polymerase is the target of the antimicrobial rifampin,
▪ Rifampin blocks the initiation of transcription.
• The process begins when the sigma factor recognizes

the promoter and binds tightly to this site.
• Sigma factors are activator and repressor proteins that control
expression of a gene or an operon.
• Sigma factors bind to these promoters to provide a
docking site for the RNA polymerase.
• Once the RNA polymerase has bound to the

appropriate site on the DNA, RNA synthesis proceeds
• Once an entire gene or group of genes (operon) has

been transcribed, the RNA polymerase dissociates from
the DNA,
• a process mediated by signals within the DNA
Translation
• Translation is the process by which

the language of the genetic code in
the form of mRNA is converted
(translated) into a sequence of
amino acids, the protein product.
• Each amino acid word is written as

sets of three nucleotides known
as codons
• Translation of mRNA occurs

simultaneously with transcription
Translation
• Translation is the protein synthesis.
• Bacteria activate the 20 amino acid building blocks of protein in
the course of attaching them to specific transfer RNA molecules
(tRNA).
• The aminoacyl-tRNAs are brought to the ribosomes by soluble
protein factors, and
• In ribosomes, the amino acids are polymerized into polypeptide
chains
• The tRNA is released from the ribosome to return for another
aminoacylation cycle.
• Many antimicrobial agents derive their selective toxicity for

bacteria from the unique features and proteins of the prokaryotic
translation apparatus
• Such as: Aminoglycosides, tetracyclines, Chloramphenicol
Control of gene expression
• The operon is regulated both negatively and positively
• A specific regulator protein or transcription factor

(repressor) encoded by regulatory gene can bind to an
operator, preventing transcription
• Negative control,
•
• The lac operon consists of three genes: lacZ, lacY, and lacA.
• Negative control is brought about by the lac repressor, which is the product
of the lacI gene.
• The operator is the negative regulatory site bound by lac repressor
• Positive control results from the action of CAP (catabolite activator

protein
• The CAP binding site is positive regulatory site
• When CAP binds the CAP site it promotes transcription
•A) Negative control of an inducible gene:
• In the absence of inducer, the repressor protein blocks transcription.
• In the presence of inducer prevents the repressor from binding DNA, and
transcription occurs.
•B) Negative control of a repressible gene:

• In the absence of corepressor, the repressor is unable to bind DNA, and
transcription occurs.
• in the presence of corepressor, the corepressor is bound to the
repressor, the repressor is able to bind DNA and transcription is blocked.
Bacterial regulatory proteins:
•C) Positive control of an inducible gene:

• In the presence of inducer, the activator protein (CAT) is
able to bind DNA and activate transcription.
•D) Positive control of a repressible gene

• In the absence of inhibitor, the activator binds DNA and
promotes transcription.
• In the presence of inhibitor, the activator is unable to bind
DNA; this inhibits transcription
Gene Expression in Microbes
➢ Genes can be turned on and off.
➢ Many bacterial genes are controlled at multiple levels and by multiple

methods.
➢ Bacteria might produce more than six different sigma factors to provide global
regulation in response to stress, shock, starvation, or to coordinate production
of complicated structures such as flagella.
➢ In a process called quorum sensing, when a sufficient number of

bacteria are present and producing a specific small molecule, virulence
and other genes are turned on
➢ Understanding of how microbial genes are expressed (turned on and off) can help
us control disease-causing bacteria.
From the Virtual Microbiology Classroom on ScienceProfOnline.com

Using Gene Expression to Control Disease
Staphylococcus & Antibiotic Resistance
• Many strains of Staphylococcus are now

resistant to penicillin.
• One bacterial protein that confers penicillin

resistance is called beta-lactamase (enzyme
that cuts up and deactivates penicillin).
• Gene for beta-lactamase only expressed in the

presence of penicillin.
• When the bacteria is not exposed to penicillin,

that gene is turned off and no beta-lactamase is
made.
• Understanding how beta-lactamase gene is

turned on/off, can help us to design a drug to
disable that gene’s expression (turn off the
gene).
MUTATIONS
• Mutations are stable hereditary changes in coding
sequences of DNA
• Mutations occur spontaneously or are induced by

different mutagens such as radiation, chemicals,
viruse
• Changes in base sequence of DNA can be:

– Harmful
– Lethal
– Helpful
– Silent
Various kind of mutations:
Transitions: replacement of a purine base with
another purine or replacement of a pyrimidine
with another pyrimidine
Transversions: replacement of a purine with a
pyrimidine or vice versa.
Deletion: Mutations result in missing DNA.
Insertions: Mutations result in the addition of
extra DNA
Inversions: An entire section of DNA is
reversed.
Mutations
• Point Mutation: A point mutation is a simple
change in one base of the gene sequence
• Frame-shift mutation: One or more bases are
inserted or deleted. Resulting a change to a
gene’s reading frame
– This type of mutation can make the DNA

meaningless and often results in a
shortened protein.
Frame-shift mutation
• Null mutation: completely destroy gene
function when there is an extensive
insertion, deletion or gross rearrangement
of the chromosome
• A nonsense mutation changes a
codon (encoding an amino acid) to a
stop codon (e.g., TAG [thymidine-
adenine-guanine]),
• which will cause the ribosome to
fall off the mRNA and end the
protein prematurely.
• A silent mutation is a change at the

DNA level that does not result in any
change of amino acid in the encoded
protein.
– This type of mutation occurs
because more than one codon may
encode an amino acid.
•
• A missense mutation: just one
different amino acid formed-caused
from a base substitution, single base
is replaced with a different one.
• The resulting protein may be

enzymatically inactive
• but this may be a conservative

mutation if the new amino acid
has similar properties (e.g., valine
replacing alanine).
Mutations
Repair Mechanisms of DNA
A number of repair mechanisms have evolved in bacterial cells to minimize

damage to DNA.
•Direct DNA repair is the enzymatic removal of damage,

• such as pyrimidine dimers and alkylated bases are removed by PHOTOLYASE
• Mismatch repair: DNA poymerase III (including proofreading

exonuclease) reduces misincorporation from per 1 change/10e8 to 1
change/10 e10-10e11
•Excision repair is the removal of a DNA segment containing the damage,

followed by synthesis of a new DNA strand.
Repair Mechanisms of DNA
• Recombinational or postreplication repair is the retrieval of
missing information by genetic recombination when both DNA
strands are damaged.
• The SOS (Save our Ship) response is the induction of many genes
(approximately 15) after DNA damage or interruption of DNA
replication.
• Error-prone repair is the last resort for DNA repair, before a

bacterial cell dies.
– It is used to fill in gaps with a random sequence when a DNA template is not
available for directing an accurate repair.
TRANSPOSABLE ELEMENTS
• Transposible elements (transposons, jumping genes) are mobile genetic
elements that can transfer DNA within a cell, from one position to another in
the genome, or between different molecules of DNA (e.g., plasmid to
plasmid or plasmid to chromosome).
Mobile genetic elements

Bacteriophages /Prophage
Plasmids
Transposons
Integrons
• Transposons are present in prokaryotes and eukaryotes.
• All transposable elements contain common elements.
• These include inverted repeats (IRs) at the ends of the elements and
• a transposases gene
Transposons (Tn)
• The simplest transposons are called insertion sequences
• IS elements encode only proteins for their own transposition
• Insertion of IS elements into a gene causes mutation
• IS elements are components of transposons
Complex (composite) transposons carry other genes, such as genes

that provide resistance against antibiotics.
• Some pathogenic bacteria use a transposon-like mechanism to
coordinate the expression of a system of virulence factors.
– The genes for the activity may be grouped together in
a pathogenicity or virulence island
Transposon
Transposable elements
• Main roles:
– Cause deletion and inversions of DNA
sequences (internal or biological
mutagenic agents)
– Insert into gene and inactivate those
genes
– Spread antibiotic resistance genes
The mobile genetic elements are responsible for the major part
of genetic variability in natural bacterial populations
Integrons are genetic elements that capture and express gene
cassettes.
Integron has three main components: an integrase gene
under the control of its own promoter (P int ), an attI site for
integration of the gene cassette, and a promoter to express
the gene cassette (P c ).
PLASMID
Plasmids are extrachromosomal genetic elements capable of
autonomous replication in bacteria
An episome is a plasmid that can integrate into the bacterial
chromosome, such as E.coli F plasmid
Most plasmids are circular double-stranded DNA molecules-
but some are linear
Plasmids provide some additional properties in bacteria
harboring them:
F: fertility factor
R: antibiotic resistance
Col: colicin production
Virulance plasmids
Ent: Enterotoxin production
Hly:Hemolysin production
CFA-I, CFA-II: Adhesion production
Plasmids
Conjugative plasmids: They are able to bring about
their own transfer from one cell to another.
Nonconjugative plasmids: They are not able to

bring their transfer from one cell to another
Without selection pressure, plasmids may be lost

Bacteriophages
• Bacteriophages ( phages ) are obligate intracellular parasites that
multiply inside bacteria by making use of some or all of the host
biosynthetic machinery .
• They are viruses that infect bacteria.
• Nucleic acid: either DNA or RNA but not both

– ds DNA, ss RNA, ss DNA
• Protein: function in infection and protect the nucleic acid

Structure of Bacteriophage
• different sizes and shapes
icosahedral
filamentous
BACTERIOPHAGES
• Virulent (lytic) bacteriophage: a
bacteriophage which always causes the
lytic cycle, resulting in a death of cell and
production new phage particles
• Temparate (or lysogenic) bacteriophage: a

bacteriophage whose DNA integrates into
host cell DNA, but doesn’t cause the lysis
of the cell.
• The integrate phage’s DNA is called

prophage
• A bacterial cell with prophage is called

lysogenic cell
BACTERIOPHAGES
• Lysogenic conversion is a state when bacterial

cell exhibit new properties, that are coded by
prophage gene
• Examples:
– Toxigenic (tox+) and nontoxigenic strains of Corynebacterium diphtheriae
– Production of erythrogenic toxin by Streptococcus pyogenes
– Production of botulinal toxin by Clostridium botulinum

Application of Bacteriophage
• Used in treatment of bacterial infections
• Used for the identification of pathogenic
bacteria (phage typing)
• Used in molecular biology
GENETIC RECOMBINATION
• Production of a unique DNA

molecule by joining together two
or more DNA fragments not
normally associated with each
other
• DNA fragments are usually

derived from different biological
sources
• The transferred donor DNA may be

integrated into the recipient's
chromosome by various mechanisms
(homologous, non-homologous).
Recombination
• Homologous recombination- occurs between closely related DNA sequences and
generally substitutes one sequence to another.
Site-Specific Recombination/Non-homologous recombination: relies on only

limited DNA sequence similarity
• This recombination is particularly important in the integration of virus

genomes into host chromosomes.
• The transfer of genetic matarials
between prokaryotes.
• Vertical gene transfer – organisms replicate their
genomes and provide copies to descendants. Passing on
genes to descendants.
• Horizontal gene transfer – donor gives part of genome to

recipient that are not descendants; three types:
– Transformation
– Transduction
– Bacterial Conjugation
Gene Transfer in Bacteria
• Horizontal gene transfer

• Three ways:
– Transformation: genes transferred from one bacterium
to another as “naked” DNA
– Conjugation: plasmids transferred from 1 bacterium
to another via a pilus
– Transduction: DNA transferred from 1 bacterium to
another by a virus
TRANSFORMATION
Transformation is a process by which a DNA fragment from a

dead, degraded bacterium enters a competent recipient
bacterium and it is exchanged for a piece of the recipient's
DNA.
CONJUGATION
Bacterial conjugation is a transfer of DNA from a
living donor bacterium to a recipient bacterium.
Secretion systems or sex pili form bridges
between cells
The 3 conjugative processes
I. F conjugation
II. Hfr conjugation
III. Resistance plasmid conjugation
F-Conjugation in E. coli
Hfr Conjugation continued…
Conjugation continued…
Transduction
• Transduction: a DNA fragment is

transferred from one bacterium to another
by a bacteriophage.
– Bacteriophage (phage) are obligate intracellular

parasites that multiply inside only living
bacteria
Transduction
• There are two types of transduction:

• – generalized transduction: A DNA
fragment is transferred from one bacterium
to another by a lytic bacteriophage
• – specialized transduction: A DNA fragment

is transferred from one bacterium to
another by a temperate bacteriophage
Transduction by a Bacteriophage
References
70
INTRODUCTION OF MYCOLOGY
PROF DR RIZA DURMAZ

YİÜ 2022
INTRODUCTION
• Mycology is the study of fungi

• Approximately 80.000 species of fungi have been described,
• fewer than 400 are medically important, and
• Less than 50 species cause more than 90% of the fungal
infections of humans and other animals.
• Fungal infections are mycosis
• Most species of fungi are beneficial to humankind.

Introduction-2
• All fungi lead a heterotrophic existence as
– Saprobes: organisms that live on dead or decaying matter
– Symbionts: organisms that live together and in which the
association is of mutual advantage
– Commensals: organisms living in a close relationship in which one
benefits from the relationship and the other neither benefits nor
is harmed
– Parasites: organisms that live on or within a host from which they
derive benefits without making any useful contribution in return;
in the case of pathogens, the relationship is harmful to the host.
Beneficial Effects of Fungi
1. Decomposition - nutrient and carbon recycling.
2. Biosynthetic factories. The fermentation property is
used for the industrial production of alcohols,
fats, citric, oxalic and gluconic acids.
3. Important sources of antibiotics, such as Penicillin.
4. Model organisms for biochemical and genetic
studies.
Eg: Neurospora crassa, Saccharomyces cerviciae.
5. Yeasts provide nutritional supplements such as
vitamins and cofactors.
General properties of fungi:
• 1. They are eukaryotic; cells contain membrane bound cell organelles
including
– nuclei, mitochondria, golgi apparatus, endoplasmic reticulum, lysosomes
– Linear chromosomes
– They have 80S ribosomes.
– They lack chlorophyll
General properties of fungi cont..
• 2. They have ergosterol in their membranes rather than cholesterol
– Ergosterol is the site of action of antifungal drugs, amphtericin B &
azole group
• 3. They have a rigid cell wall and are therefore non-motile,

– All fungi possess cell wall made of chitin not peptidoglycan like
bacteria.
– Thus fungi are insensitive to antibiotics as penicillins.
– Some yeast and molds have melanized cell walls, imparting a brown
or black pigment.
• Such fungi are dematiaceous.
• The term "dematiaceous" refers to the characteristic dark appearance of this
group of fungi as it grows on agar
The fungal cell wall
• Chitin is a polysaccharide composed of long
chain of n-acetyleglucasamine.
• Chitin synthesis is inhibeted by Nikkomycin
• The fungal cell wall contain other

polysaccharide, -glucan, which is the site of
action of some antifungal drugs.
• These antifungal drugs are known as the echinocandins.

– Caspofungin
Micafungin
Anidulafungin
– They block fungal cell wall synthesis by

inhibiting 1,3-beta glucan synthase
•
- D-Glucan Testing Is Important for
Diagnosis of Invasive Fungal Infections
• Mannoproteins are composed of mannose-based polymers (mannan)
found on the cell surface
– Antibodies are readily developed against these molecules on the cell
surface.
– The potential variations in the composition and linkages of the mannan side chains
allow fungi to avoid easy immune detection by an infected host.
• The measurement of galactomannans (GM), a [1-3]-β-D-glucan (BD) has been
widely used as a biomarker for early diagnosis of invasive pulmonary aspergullosis
(IPA) in neutropenic patients, together with polymerase chain reaction (PCR) assay.
• GM and BD are polysaccharide of the Aspergillus wall that can be detected during
the invasive disease in different body fluids (e.g., serum, BAL and cerebrospinal
fluid).
• 4.Fungi may be unicellular or
multicellular
– Yeasts are usually unicellular.
– Molds are multicellular organisms

consisting of threadlike tubular
structures, called hyphae
• Hyphae are either coenocytic
(hollow and multinucleate) or
septate (divided by partitions
or cross-walls)
– The hyphae form together to
produce a mycelium
Fungal hyphae form a network
called a mycelium (pl. mycelia)
Mycelium
Hypha (plural hyphae)

• 5.They are chemoheterotrophs (require organic compounds for both
carbon and energy sources)
• 6. Fungi are osmotrophic; they obtain their nutrients by absorption.

Grow at lower pH-5 than bacteria
Grow in high salt and sugar
• 7. They obtain nutrients as saprophytes (live on decaying matter) or

as parasites (live on living matter).
• 8. Most fungi exhibit aerobic respiration, although some are

facultatively anaerobic (fermentative), and others are strictly
anaerobic
Common Fungal culture media: There are selective and non-selective media
SDA: Sabouraud’s dextrose agar
PDA: Potato Dextrose Agar,
BHIA: Brain-heart infusion agar
IMA: Inhibitory mold agar
DTM: Dermatophyte test medium
CHROMagar medium
• 9. Fungi may reproduce by either asexual or sexual process.
• The asexual form is called the anamorph,

– Asexual spores consist of two general types:
sporangiospores and conidia.
• The sexual form is called the teleomorph,

– Its reproductive structures are called spores
• ascospores,
• zygospores,
• basidiospores…...
• Fungi reproduce by emission of spores.
– Both sexual and asexual reproduction are present in them.
• Sexual: involving meiosis,
– the haploid nuclei of donor and recipient cells fuse to
form a diploid nucleus, which then divides by classic
meiosis
• Asexual reproduction: involving mitosis only

– involves mitotic division of the haploid nucleus and
– is associated with production by budding, spore-like
conidia or, alternatively by the separation of hyphal
elements.
6.03.2023 15
• 10. They grow either reproductively by budding or non-reproductively by
hyphae tip elongation.
• 11. Food storage is generally in the form of lipids and glycogen.
• 12. Some fungi such as Cryptococcus and yeast form of Histoplasma

capsulatum possess polysaccharide capsules that help them to evade
phagocytosis
Natural habitat
• Environment: Many fungi live in the soil (Geophilic) and are
occasional pathogens of both animals and humans
• Animals: Zoophilic fungi live in the hair and skin of animals

but can be transmitted to humans
• Humans: Some yeast line on or within human body as a part

of normal human flora
– Candida albicans
Classification of fungi:
• Fungi are classified:
– Based on mode of spore production
– Based on morphology of the thallus (vegetative
structure).
Recently, ultrastructural features, biochemical, and molecular

characteristics are brought to bear, often resulting in changes
in the original taxonomic designation
Schematic diagram of the fungal rDNA gene cluster .
Schematic diagram of the fungal rDNA gene cluster.

Genes encoding 18S, 5.8S and 28S ribosomal RNA subunits are separated by the internal
transcribed sequences 1 (ITS1) and 2 (ITS2
Major phyla of fungi causing disease in
humans
• Phylum Glomeromycota (Mucormycetes)
• Phylum: Basidiomycota (Basidiomycetes)
• Phylum: Ascomycota (Ascomycetes)
– Class: Pneumocystidomycetes
– Class: Saccharomycetes
– Class: Eurotiomycetes
– Class: Sordariomycetes
• Phylum: Microspora (Microsporidia)

1-Mucormycetes
(Formerly Zygomycetes)
• They are largely saprophytic fungi
• Hyphae are mostly aseptate
– There is an extensive coenocytic
mycelium
• They have both asexual and sexual
spores
• Asexual spores - Sporangiospores:
present within a swollen sac- like
structure called Sporangium
• Examples: Rhizopus, Mucor

Mucormycetes cont..
• Sexual spores –Zygospore
Sexual reproduction in Rhizopus nigricans.
– (a) Plus and minus spores are planted
on a nutrient medium.
– (b) The mycelium expands, and plus

and minus filaments become closely
associated.
– (c) Fusion produces a zygospore,
– (d) Zygospore germinates to produce a

new hypha.
2- Basidiomycetes
– Most members of the Basidiomycetes have filamentous form, but some
are typical yeasts.
– Hyphae have complex septa.
– Sexual reproduction results in four progeny basidiospores supported

by a club-shaped basidium
– Asexual: production of conidia by budding from a mother cell

or within a hyphal fragment
– Examples: Cryptococcus, Malassezia, and Trichosporon

3. Ascomycetes
• The phylum Ascomycota contains almost
50% of all named fungal species and
accounts for approximately 80% of fungi
of medical importance.
➢ Includes both yeasts & filamentous fungi
➢ Asexual spores are called conidia borne
on conidiophore
• Sexual spores called ascospores are present
within a sac like structure called Ascus.
– Each ascus has 4 to 8 ascospores
➢ They have narrow, septate hyphae
– Examples:
• Histoplasma,
• Aspergillus,
• Blastomyces,
• Dermatophytes
• .
• Different types of Conidia
– Microconidia are small,
– Macroconidia are large or multicellular
Microconidia and characteristic
macroconidia
Conidia cont..
• A) Chlamydoconidia (or Chlamydospore) : Large, thick-walled, usually
spherical conidia produced from terminal or intercalary hyphal cells.
– terminal
intercalary
– They are produced by Trichophyton schoenleinii and T. verrucosum

– They are thick walled cells that are larger than other cells and
arranged singly or in groups.
Conidia cont..
B)Arthrospores (or arthroconidia) : Conidia that result from the
fragmentation of hyphal cells
Trichosporon beigeilli and Coccidioides immitis
C) Blastoconidia (blastospores): Conidial formation through

a budding process (eg, yeasts,)
Saccharomyces. Budding yeast cells or

blastoconidia. Conjugating blastoconidia.
Conidia cont..
• D) Phialoconidia: Conidia that are produced by
a “vase-shaped” phialide (eg, Aspergillus
fumigatus
Phialides form on top of swollen vesicle at

the end of a long conidiophore.
Class in Ascomycetes
• The Phylum Ascomycota consists of four
classes of medical importance:
– Class: Pneumocystidomycetes
– Class: Saccharomycetes
– Class: Eurotiomycetes
– Class: Sordariomycetes
Pneumocystidiomycetes
• Pneumocystidiomycetes had formerly been considered
protozoan.
– Human-derived strain is Pneumocystis jirovecii.
– The organism exists in a vegetative form that reproduces

asexually by binary fission.
– Sexual: Fusion of compatible mating types results in a

spherical cyst or spore case, which on maturity contains
eight spores.
Saccharomycetes
• They contain the ascomycetous yeasts, which are
characterized by vegetative yeast cells that proliferate by
budding or fission
– Asexual: production of blastoconidia by budding from a
mother cell.
– Sexual: either not seen or by conjugation between two

single cells or by "mother-bud" conjugation
• Examples: Candida and Saccharomyces

Eurotiomycetes
• Sexual reproduction leads to the formation of ascospores.
• This class has seven orders that include species pathogenic to

humans.
Among the more important are
– the dermatophytes
– dimorphic systemic pathogens
• (including H. capsulatum and Blastomyces dermatitidis),
– Aspergillus and Penicillium.
• Sordariomycetes contain
– genus Fusarium, Scedosporium
– the teleomorphs of numerous melanized

(dematiaceous) fungi of medical importance
Microspora (Microsporidia)
• Microsporidia are
– obligate intracellular,
– unicellular,
– sporeforming eukaryotes.
• Presently, human infections have been shown

to involve nine different genera ( such as Anncaliia,
Encephalitozoon, Endoreticulatus, Enterocytozoon, Nosema, Pleistophora, Vittaforma,
Tubulinosema, and Trachipleistophora)
• Fungi Imperfecti:
• In some fungi, the sexual stage, or teleomorph,
has disappeared or has not yet been
discovered.
• In the past, these asexual fungi were classified

in an artificial group, the "Fungi Imperfecti"
(form-division Deuteromycota).
Fungi Classification based on
Morphology:
• Fungi exist in two fundamental forms; the filamentous (hyphal) and single celled
budding forms (yeast). But, for they are studied as moulds, yeasts, yeast like and
dimorphic fungi.
• 1. Moulds (Molds): Filamentous fungi

– Eg: Aspergillus sps, Trichophyton rubrum
• 2. Yeasts: Single cells, usually spherical to elipsoid in shape,

– Eg: Cryptococcus neoformans, Saccharomyces cerviciae
• 3. Yeast like: Similar to yeasts but produce pseudohyphae

– Eg: Candida albicans
• 4. Dimorphic: Fungi existing in two different morphological forms at two different

environmental conditions.
Yeasts:
• Yeasts are unicellular spherical to ellipsoid cells.
• They reproduce by budding, which result in
blastospore (blastoconidia) formation or fission.
Yeast
• Some yeast such as Cryptococcus and the yeast
form of Blastomyces dermatatidis produce
polysaccharide capsule.
– Capsules can be demonstrated by negative staining
methods using India ink or Nigrosin.
• On culture, they produce smooth, creamy colonies

Yeast colonies
Yeasts colonies resemble bacterial colonies in
appearance and in consistency.
Mucoid
colonies
Candia albicans also has the
ability to produce germ
tube.
Candida Colonies
Dimorphic fungi
• Fungi existing in two different morphological forms at two
different environmental conditions.
– They exist as yeasts in tissue and in vitro at 37 °C and as moulds in

their natural habitat and in vitro at room temperature.
• Other conditions associated with dimorphism: iron limitation, pH

changes, elevated levels of CO2 ,serum, quorum sensing molecules,
nutrient availability and hypoxia all regulate morphogenesis
• Dimorphism in fungi is reversible
– Eg: Histoplasma capsulatum,

– Blastomyces dermatidis,
– Paracoccidiodes brasiliensis,
– Coccidioides immitis
Moulds (molds)/Flamentous fungi:
• The thallus of mould is made of hyphae, which are cylindrical
tube like structures that elongates by growth at tips.
– Thallus is body or vegetative structure of a fungus
• Fungal hyphae form a network called a mycelium (pl. mycelia)
• The hyphae may be branched or unbranched.
• The hyphae may be septate or aseptate.

Septate hyphae - cross walls that divide them into unicellular units
• Pores to allow cytoplasm & nuclei to pass
– Coenocytic hyphae- no septa, continuous cells with many nuclei
mycelium: septate mycelium: non septate
• Mycelia are of three kinds:
• 1. Vegetative mycelia are those that penetrates the surface of the medium and
absorbs nutrients.
• 2. Aerial mycelia are those that grow above the agar surface
• 3. Fertile mycelia are aerial hyphae that bear reproductive structures such as
conidia or sporangia.
• Colony Morphology of molds
The colonies formed by molds are often described as filamentous, hairy, or
woolly
Fungi are slow growing, with cell doubling times in terms of hours rather than
minutes.
Structure or appearance of hyphae
• Hyphae may have some specialized structure or appearance that
aid in identification.
• Spiral hyphae: These are spirally coiled hyphae commonly seen in
Trichophyton mentagrophytes.
• Pectinate body: These are short, unilateral projections from the hyphae
that resemble a broken comb.
• Commonly seen in Microsporum audouinii.
• Favic chandelier: These are resemble a
chandelier or the antlers of the deer (antler
hyphae).
– They occur in Trichophyton schoenleinii and
Trichophyton violaceum.
• Nodular organ: This is an enlargement in the

mycelium that consists of closely twisted
hyphae.
– Often seen in Trichophyton mentagrophytes and
Microsporum canis.
• Racquet hyphae: There is regular
enlargement of one end of each
segment with the opposing end
remaining thin.
– Seen in Epidermophyton floccosum,
Trichophyton mentagrophytes.
• Rhizoides: These are the root like

structures seen in portions of
vegetative hyphae in some members of
zygomycetes.
• Germ-tube: The initial hyphal
outgrowth of a germinating spore or
yeast; especially important for
identification of Candida albicans.
• Pseudohyphae (or
Pseudomycelium):chains of successively
budding yeast cells that have not
detached from one another.
Fungal Interactions with the
Human Host
• Fungi form an important part of human microbiome
• Fungi are ubiquitous transient or persistent human colonisers and
form the mycobiome
• These complex interactions can be viewed as a spectrum of

symbiotic relationships
– Mutualistic
– commensal
– Parasitic
Shifts in mycobiomes (dysbiosis) is associated with various

diseases.
• Fungal populations in and on
different anatomical sites.
• Fungal populations have been

identified in and on almost all
human body sites.
• This figure is a schematic

representation of the most
commonly identified fungal
genera under nonpathological
conditions
• Infect Immun 2021, 89:e00648- 20.
https://doi.org/10.1128/IAI.00648-20
Niche-specific fungal symbionts
• Fungal symbionts vary in
– route of acquisition,
– Niches/ inhabited, and
– type of symbiotic relationship formed.
• Some species are acquired at birth by direct transmission,
– leading to colonization of the skin and mucosal surfaces,
• while many fungal pathogens are ubiquitous within the environment,
– leading to constant exposure by oral or respiratory routes, as well as skin contact.
– Most of these exposures are transient,
• due to clearance by host immune responses or
• out competition for resources by the microbiome.
• changes in immune status or dysbiosis could lead to
chronic colonisation and eventual host damage.
Pathogenesis of Fungal Disease
• Primary pathogens:
– Capable of initiating infection in a normal, apparently immunocompetent
host.
– Blastomyces dermatitidis,
– Coccidioides immitis (and Coccidioides posadasii),
– Histoplasma capsulatum, and
– Paracoccidioides brasiliensis
• Opportunistic pathogens:
– The opportunistic fungal pathogens generally only cause infection when there
are disruptions in the protective barriers of the skin and mucous membranes
or when defects in the host immune system allow them to penetrate,
colonize, and reproduce in the host
– Ex: Candida spp., Cryptococcus neoformans, and Aspergillus spp.
Virulance factors
Fungal pathogens can use more than one of the following virulance factors to produce
mycosis
• Grow at 37° C
• Thermal dimorphism
• WI-1 cell Wall glycoprotein (Adhesin and stimulate TH2) (B. dermatitidis)
• Stimulation of ineffective TH2 responce (B. dermatitidis)
• Molecular mimicry (coccidioides)
• Urease and proteinase production (coccidioides)
• Resistance to phagocytic killing (coccidioides)
• Survival in macrophages (H. capsulatum)
• Modulate pH of phagosom (H. capsulatum)
• Alteration of cell Wall composition (H. capsulatum)
• Capsule (C. neoformans)
• Adherence (C. albicans)
• Bud-hyphae transition (C. albicans)
• Cell surface hydrophobicity (C. albicans)
• Mycotoxins
Mycotoxins
• Mycotoxin can be produced mainly by mycelial
structure of filamentous fungi or specifically
molds
– Mycotoxins have been implicated in a
variety of illnesses and clinical syndromes in
humans and in animals.
• These mycotoxins are secondary fungal

metabolites that cause diseases, known
collectively as mycotoxicoses, after
– ingestion,
– inhalation, or
– direct contact with the toxin.
Mycotoxicoses
• Mycotoxicoses may manifest as acute or chronic disease, ranging
from rapid death to tumor formation.
– In this regard, mycotoxicoses are analogous to the pathologies caused by
other “poisons” such as pesticides or heavy metal residues.
• The presenting symptoms and severity of a mycotoxicosis depend
on
– the type of mycotoxin;
– the amount and duration of exposure;
– the route of exposure; and
– the age, sex, and health of the exposed individual.
• In addition,
– a variety of other circumstances, such as malnutrition, alcohol abuse,
infectious disease status, and other toxin exposures, may act synergistically to
compound the effect and severity of mycotoxin poisoning.
Mycotoxins
• Mycotoxins may cause a harmful effect to animals as well as humans such as
– carcinogenic, nephrotoxicity, mutagenic, immunosuppressive, estrogenic neurotoxicity,
reproductive and developmental toxicity, hepatotoxicity and indigestion
• Mycotoxins mostly affect the public health and agro-economic significance
• Mycotoxin may become a biological weapon in bioterrorism because of its acute and
chronic toxicities
• Mycotoxins are commonly used as antibiotics and growth promotants
• Most of the mycotoxin are found as natural contaminant food, mainly in vegetable and
feed.
– Nut, cereals, oilseeds, dried fruits, spices, and food from animal origins for example
milk, egg, and meat
Mycotoxins: Different Types
• More than 300-400 mycotoxins presently identified
• There are few types of mycotoxins, which are a significant threat
to the life and health of human and live stocks such as
Aflatoxin B1 Hepato-toxic, carcinogenic
Ochratoxin A Nephrotoxic carcinogenic
Fumonisins Teratogenic Carcinogenic
Deoxynivalenol Gastroenteritis,
Zerealenone Hyperestrogenism
T2-Toxin Gastric ulcers/Enteritis
Mycotoxins
Mycotoxins
Ochratoxins
Aflatoxins
Mycotoxins are low molecular weight and thermal-stable

secondary metabolite of toxic molds that belong to genera
Aspergillus, Penicillium, Alternaria, and Fusarium.
Classification of Human Mycoses
• Superficial:
– The superficial mycoses are limited to the
very superficial surfaces of the skin and hair
• Cutaneous:
– Cutaneous mycoses are infections of the
keratinized layer of skin, hair, and nails
– Infections of the skin involving
Dermatophytes are called dermatophytoses
• Subcutaneous mycoses:
– Subcutaneous mycoses involve the deeper
layers of the skin, including the cornea,
muscle, and connective tissue
Classification of Human Mycoses
Endemic mycoses/ systemic mycoses
– The endemic mycoses are caused by the classic
dimorphic fungal pathogens
– These fungi are generally confined to geographic
regions in which they occupy specific
environmental or ecologic niches.
Opportunistic mycoses
– The opportunistic mycoses are
infections attributable to fungi that are
normally found as human commensals
or in the environment
CLASSIFICATION OF HUMAN MYCOSES
• Fungal infections (Mycosis) may be classified according to the tissues
infected, as well as by specific characteristics of organism groups.
Summary of fungi associated with human disease
Summary of fungi associated with human disease (cont..)
Specimen Collection and Processing
• Clinical samples:
– Blood,
– CSF,
– Hair,
– Skin scraping,
– corneal scraping
– Nail,
– Biopsy,
– Vaginal and urethral discharge
Laboratory Diagnosis of Fungal Diseases
• Conventional Microbiologic Methods

– Direct microscopy (Gram, Giemsa, and calcofluor white stains)
– Culture (Sabouraud dextrose, brain heart infusion, CHROMagar )
– Identification
– Susceptibility testing
• Histopathologic Methods
– Routine stains (H&E)
– Special stains (GMS, PAS, Mucicarmine)
– Direct immunofluorescence
– In situ hybridization
GMS, Gomori methenamine silver; H&E, hematoxylin and eosin; PAS, periodic acid–Schiff.
Laboratory Diagnosis of Fungal Diseases
• Immunologic Methods
– Antibody
– Antigen
• Molecular Methods
– Direct detection (nucleic acid amplification)
– Identification
– Strain typing
• Biochemical Methods
– Metabolites
– Cell wall components
– Enzymes
Sites of action of antifungals.
References
• Murray et al. Medical Microbiology, 2021
• Sabuncuoglu S, Mycotoxins and Food safety, 2019
74
General Virology
Prof Dr Rıza DURMAZ-2023
Viral Properties
 Viruses are non-living entities
 Viruses are inert filterable agents
 Viruses are obligate intracellular parasites
 Viruses cannot make energy or proteins independent of
a host cell
 Viruses must be able to use host cell processes to produce their components
(viral messenger RNA, protein, and identical copies of the genome).
 Viruses must encode any required processes not provided by the cell.
 Viral genome are RNA or DNA but not both.
 Viruses do not have the genetic capability to multiply by
division
Size of Viruses
A small virus has a diameter of

about 20 nm.
Parvovirus
A large virus have a diameter of
up to 400 nm.
Poxviruses
Shape of Viruses
 Spherical
 Rod-shaped
 Brick-shaped
 Tadpole-shaped
 Bullet-shaped
 Filament
Structure of Viruses
Virion Structure
✓ The complete infectious unit of virus particle
✓ Structurally mature, extracellular virus particles
✓ Viruses have a naked capsid or envelope with attached
proteins
Lipid Envelope Nucleic Acid
Tegument
Protein
Capsid
Core
Virion
Associated
Spike
Polymerase
Projections
❑ Viral core
❑Core of a virus is the nucleic acid (either DNA or RNA) that
makes up the viral genome
❑Control the viral heredity and variation,
❑Responsible for the infectivity.
 The genome of a virus can be either DNA or
RNA
 DNA-double stranded (ds): linear or circular
Single stranded (ss) : linear or circular
 RNA- ss: segmented or non-segmented
ss: polarity+(sense) or polarity – (non-sense)
 ds: linear (only reovirus family): (one strand + and the
second strand –) or ambisense (both + and – polarity on the
same strand).
Viral Capsid
 The protein shell, or coat, that encloses the nucleic acid
genome.
 Functions:
 a. Protect the viral nucleic acid.
 b. Participate in the viral infection.
 c. Share the antigenicity
 Surface structures are important in adsorption and penetration

The surface structures of the capsid and envelope mediate the
interaction of the virus with the target cell through a viral
attachment protein (VAP) or structure.
Removal or disruption of the outer package inactivates the virus.
Antibodies generated against the VAP prevent virus infection.
Capsomere or morphologic subunits
Capsomere: subunit of the capsid

 Smallest morphological unit visible with an
electron microscope
 A capsomere is generally composed of either five or six

individual protein molecules, each one referred to as a
structural subunit, or protomer.
Viral capsid and capsomer
envelope
Capsid
Capsomere
Viral core
Viral Capsomere
 Their major puposes are

 To facilitate transfer of the viral NA from one host to another
 To protect viral genom against inactivation
 To participate in the attachment of the virus to the susceptible
cells
 To determine antigenic characteristics of the virus
 To provide the structural symmetry of the virus particle.
Symmetry of Nucleocapsid
 Cubic /Icosahedral  Helical
Complex
Eg:
Hepatitis viruses Eg:
Influenza Virus
Herpesviruses Measles Virus
Polio virus Mumps Virus
Rhinovirus Parainfluenza Virus
Rubella Virus Rabies Virus
Respiratory Syncytial
HIV Virus(RSV
Adenovirus
Symmetry types
 Rod shaped viruses have helical symmetry
 Spherical shaped viruses have icosahedral symmetry
 Nonsymmetric capsids are complex forms and are

associated with certain bacterial viruses
Schematic drawing of two basic types of virions,
naked capsid virus and enveloped virus
Nucleocapsid
 The core of a virus particle consisting of the genome plus

a complex of proteins.
 Complex of proteins = Structural proteins +Non- Structural
proteins (Enzymes &Nucleic acid binding proteins)
 Viral Enzyme:
 Lysozyme : bacteriophage genomes encode lysozyme
 Makes hole in cell wall
 Lyses bacterial cell
 Nucleic acid polymerases: RNA polymerase, Reverse transcriptase
 Neuraminidases (example: Influenza virus)
 Enzymes that cleave glycosidic bonds
 Allows liberation of viruses from cell
Integrase catalyzes the integration of virally derived DNA
into the host cell DNA in the nucleus.ex:Retrovirus (HIV..)
NS3 Serine protease processes the polyprotein precursor to
multiple functional proteins, ex: HCV
Envelope
 The envelope is a membrane composed of lipids, proteins, and
glycoproteins
 It has a membrane structure similar to cellular membranes.
 Cellular proteins are rarely found in the viral envelope
 Envelope surrounds some viral particles.

 All of the negative-strand RNA viruses are enveloped
 It is acquired during viral maturation by a budding process through a

cellular membrane,
 Viruses-encoded glycoproteins are exposed on the surface of the

envelope.
 Glycoproteins are called “spikes” or “peplomers” or “viral
envelope proteins.”
 Some glycoproteins act as VAPs, capable of binding to structures

on target cells.
 VAPs that also bind to erythrocytes are termed hemagglutinins
(HAs).
 Some glycoproteins have other functions, such as the
 Neuraminidase (NA) of orthomyxoviruses (influenza)
 Fc receptor and the C3b receptor associated with herpes simplex virus
(HSV) glycoproteins, or
 the fusion glycoproteins of paramyxoviruses.
 Glycoproteins, especially the VAPs, are also major antigens that elicit
protective immunity.
SARS-CoV-2 spike proteins
Functions of envelope
 Antigenicity: Some viruses possess
neuraminidase and hemagglutinin
 Infectivity: Attachment, fussion, elution of
the viruses
Influenza virus:
HA: attachment,
NA: elution (release
of virus),
gp40: fussion
Comparison of naked and enveloped virus particles.
Nucleocapsid Envelope
Capsid
Nucleic
acid Nucleic
acid
Capsid
(composed of
capsomeres)
Naked virus Enveloped virus

Properties of Enveloped Viruses
They are environmentally labile—disrupted by the following:

 Acid
 Detergents
 Drying
 Heat
 Must stay wet
 They are released by budding and cell lysis
 Generly, they cannot survive the gastrointestinal tract
 They spread in large droplets, secretions, organ transplants, and blood
transfusions
 They elicit hypersensitivity and inflammation to cause
immunopathogenesis
Properties of Naked virus
 They are environmentally stable to the following:
 Temperature
 Acid
 Proteases
 Detergents
 Drying
 They are released from cell by lysis
 They can be spread easily (on fomites, from hand to hand, by dust, by small
droplets)
 Many of these viruses are transmitted by the fecal-oral route and can
endure transmission even in sewage
 They can survive the adverse conditions of the gut
 They can be resistant to detergents and poor sewage treatment
Classification of Viruses
CLASSIFICATION OF VIRUSES
-------basis of classification
 Virion morphology: Size, shape, type of symmetry, presence or
absence of envelope
 Physicochemical properties of the virion: molecular mass, pH
stability, thermal stability, susceptibility to physical and chemical
agents, especially ether and detergent
 Virus genome properties
 Virus protein properties: number, sizes and funcional activities
 Genome organization and replication
 Antigenic properties
 Biologic properties: natural host range, mode of transmission,
pathogenicity, tissue tropism, and pathology
 Disease: encephalitis and hepatitis viruses, for example
BASIC PROPERTIES OF DNA VIRUSES
• Most DNA viruses
• are double-stranded (dsDNA)
• have icosahedral symmetry
• replicate in the nucleus
• are linear
• Exceptions
• parvoviridae
• only 1 strand of DNA (ssDNA)
• poxviridae
• no icosahedral symmetry
• surrounded by complex structural proteins
• replicates in cytoplasm
• papilloma, polyoma, and hepadna
• are non-linear (circular)
•
BASIC PROPERTIES OF RNA VIRUSES
• Most RNA viruses
• are single-stranded (ssRNA)
• have helical symmetry
• replicate in the cytoplasm
• are enveloped
• Exceptions
• double-stranded (dsRNA)
• reoviruses
• icosahedral
• Reoviruses, picornaviruses, togaviruses, flaviviruses,caliciviruses
• replicate in nucleus
• Retroviruses and orthomyxoviruses
• non-enveloped
• Picornaviruses, caliciviruses,reoviruses, hepeviruses
BASIC PROPERTIES OF RNA VIRUSES
 RNA viruses replicate their genomes using virally encoded RNA-
dependent RNA polymerase (RdRp).
 Subgroups of RNA Viruses
 Positive Strand RNA Viruses
 Negative Sense and Ambisense RNA Viruses
 An ambisens genome is a genome which both nucleic acid
strands encode for proteins
 dsRNA Viruses
➢ Positive-strand RNA viruses of animals also use a common strategy to
express RdRp.
➢ RdRp is not found within the assembled virion.
➢ Instead it is translated directly from the infecting genome shortly after
penetration.
➢ RdRp and other viral proteins are encoded as a polyprotein that is
cleaved by virally encoded proteases.
An ambisens genome is a genome which both nucleic acid strands encode for proteins .
This expression strategy is found in four genera of segmented negative stranded RNA viruses:
Arenavirus, Phlebovirus, Tospovirus, and Tenuivirus
(+) polarity RNA viruses (-) polarity RNA viruses
Viral (parental) RNA acts as a mRNA Cannot use their parental RNA as a mRNA
Have no RNA polymerase Have to synthesize a (+) pozitif RNA
with the help of RNA polymerase enzyme
dsRNA Viruses
 Family Reoviridae is a large family of dsRNA

viruses. (ex. Rotavirus
 Reoviruses also have segmented genomes,

11-12 segments of dsRNA.
 Reoviruses are nonenveloped and particles

consist of two or three concentric icosahedral
capsid layers.
 DNA viruses associated with human disease are
divided into 7 families.
 The RNA viruses may be divided into at least 13

families
The viral families are determined by the structure of the
genome and the morphology of the virion. SS: single
strand DNA
The RNA viruses, their genome structure, and their
morphology. E, Enveloped;N, naked capsid.
Families of DNA Viruses and Some Important
Members
Family Members*
Poxviridae Smallpox virus, vaccinia virus, monkeypox,
canarypox, molluscum contagiosum
Herpesviridae Herpes simplex virus types 1 and 2, varicella-zoster
virus, Epstein-Barr virus, cytomegalovirus, human
herpesviruses 6, 7, and 8
Adenoviridae Adenovirus
Hepadnaviridae Hepatitis B virus
Papillomaviridae Papilloma virus
Polyomaviridae JC virus, BK virus, SV40
Parvoviridae Parvovirus B19, adeno-associated virus
Families of RNA Viruses and Some Important Members
Family* Members†
PARAMYXOVIRIDAE Parainfluenza virus, Measles virus, Mumps virus, Respiratory syncytial
virus, Metapneumovirus
ORTHOMYXOVIRIDAE Influenza virus types A, B, and C
CORONAVIRIDAE Coronavirus, severe acute respiratory syndrome
Arenaviridae Lassa fever virus, Tacaribe virus complex (Junin and Machupo
viruses), lymphocytic choriomeningitis virus
Rhabdoviridae Rabies virus
Filoviridae Ebola virus, Marburg virus
Bunyaviridae California encephalitis virus, La Crosse virus, sandfly fever virus,
hemorrhagic fever virus, Hanta virus
Retroviridae Human T-cell leukemia virus types I and II, Human immunodeficiency
virus,
Reoviridae Rotavirus, Colorado tick fever virus
Togaviridae Rubella virus;
Flaviviridae Hepatitis C virus, Yellow fever virus, Dengue virus, St. Louis
encephalitis virus, West Nile virus,
Caliciviridae Norwalk virus, calicivirus
Picornaviridae Rhinoviruses, Poliovirus, Echoviruses, Coxsackievirus, Hepatitis A
virus
Isolation of Virus
 Laboratory animals: cows; chickens; mice; rats; suckling mice
 Embryonated eggs
 Chorioallantoic membrane
 Allantoic cavity
 Amniotic cavity
 Yolk sac
 Organ/Tissue/Cell Culture:
 Primary
 Diploid cell line
 Tumor or immortalized cell line
Embryonated Hen’s Egg
 Chorioallantoic membrane (CAM)
– visible lesions called pocks. e.g.
Variola, Vaccinia virus
 Allantoic cavity – Influenza virus

(vaccine production) &
paramyxoviruses
 Amniotic cavity – primary isolation

of Influenza virus
 Yolk sac – Chlamydia, Rickettsiae &

some viruses
40 3/8/2023
Organ and tissue culture
 Primary cell cultures are obtained by dissociating specific animal organs with trypsin or
collagenase. They can be subcultured only once or twice e.g. primary monkey or baboon kidney
 Primary cells can be dissociated with trypsin, diluted, and allowed to grow into new
monolayers (passed) to become secondary cell cultures.
 Diploid cell lines are cultures of a single cell type that are capable of being passed a large but
finite number of times.
 They can be subcultured 20 to 50 times e.g. human diploid fibroblasts such as MRC-5
 Tumor cell lines and immortalized cell lines derived from tumours of human or animal tissue e.g.
Vero, Hep2
Cell Culture
 Routinely used for growing viruses
 Classified into 3 types:
 Primary cell culture – normal cells freshly taken from body &
cultured, limited growth
1. Primary Rhesus monkey kidney (PRhMK)
2. Chicken embryo fibroblast
3. Human amnion cell culture
 Diploid cell strains – cells of single type (fibroblast cells) that can be
subcultivated for limited number of times, mostly 50
1. WI-38: human embryonic lung cell
2. HL-8: Rhesus embryo cell
3. Monkey kidney cells (MKC)
 Continuous cell lines – malignant cells, indefinite subcultivtion

1. HeLa: Human Ca of cervix cell line
2. HEP-2: Human epithelioma of larynx
3. Vero: Vervet monkey kidney
4. McCoy, Detroit-6, BHK-21, Kb
Cell Culture
 Primary monkey kidney cells (MKC) are excellent for
the recovery of
 influenza viruses,
 paramyxoviruses,
 many enteroviruses, and
 some adenoviruses.
 Human fetal diploid cells support the growth of a
broad spectrum of viruses (e.g.,
 HSV, VZV, CMV, adenoviruses, picornaviruses….
 HeLa cells are excellent for the recovery of
 respiratory syncytial virus,
 adenoviruses, and
Cell Culture
 Tissues trypsin & mechanical shaking Individual cells
 Cells are washed, counted & suspended in a growth
medium.
 Growth medium – Minimum Essential Medium (MEM):

essential aminoacids, vitamins, salts, glucose & bicarbonate
in 5% CO2 with 5% fetal calf or calf serum, antibiotics &
phenol red indicator
Cell Culture Bottles / Tubes
Detection of virus growth in cell cultures
1. Cytopathic effects (CPE) –

morphological changes in cultured cells, seen under
microscope, characteristic CPE for different groups of
viruses
2. Metabolic Inhibition – no acid production in

presence of virus
3. Hemadsorption – influenza & parainfluenza

viruses, by adding guinea pig erythrocytes to the
culture
Detection of virus growth in cell cultures
4. Interference – growth of a non cytopathogenic virus can
be tested by inoculating a known cytopathogenic virus:
growth of first virus will inhibit the infection by second
5. Transformation – oncogenic viruses induce

transformation & loss of contact inhibition – microtumors
6. Immunofluorescence – test for viral Ag in cells from viral

infected cultures.
7. Hemagglutination- detect virus in cell culture medium.
8. Hemagglutination inhibition (HI: test for identification of

the virus by using the specific antibody
Detection of virus-infected host
 Inclusion Bodies:
 In the course of viral multiplication within cells, virus-specific
structures called inclusion bodies may be produced
 They become far larger than the individual viruse particle and offen
have an affinity for acid dyes (eg, eosin)
 They may be situated in the nucleus (herpesviruses), in the cytoplasm

(poxvirus), or in both (measles virus)
Normal cell and CPE
Viral Replication
Virus replication
Steps in Viral replication
 1. Recognition of the target cell
 2. Attachment
 3. Penetration
 4. Uncoating
 5. Macromolecular synthesis
 a. Early mRNA and nonstructural protein synthesis
 b. Replication of genome
 c. Late mRNA and structural protein synthesis
 d. Posttranslational modification of protein
 6. Assembly of virus
 7. Budding of enveloped viruses
 8. Release of virus
Attachment/Adsorption
 Virus attaches to the cell
surface.
 Viral attachment protein (VAP)
recognizes specific receptors
on the cell surface
 Cells without the appropriate receptors are

not susceptible to the virus.
C
A
VP1, 2, 3
R
CELL MEMBRANE
gp120
VIRUS
Hemagglutinin
S1
gD
Fibre CAR
coxsackievirus and adenovirus receptor (CAR )

PENETRATION
(Virus enters the cell)
 Virions are either engulfed into vacuoles by “endocytosis” or the

virus envelope fuses with the plasma membrane to facilitate entry
 Endocytosis eg: HPV, Adenovirus, Parvovirus
 Entry by fusing with the plasma membrane.
 Some enveloped viruses fuse directly with the plasma membrane.
 Thus, the internal components of the virion are immediately delivered to
the cytoplasm of the cell.
 Eg: HIV, Influenza virus, Rabies, Coronovirus
Internalization (PENETRATION AND
UNCOATING)
OR
After attachment, virus Whole virion is internalized
penetrates the plasma (viropexis)
membrane and releases its
genone inside cytoplasm
(fusion)
viropexis
Fusion
HIV
-entry by fussion
UNCOATING
 Uncoating is usually achieved by cellular proteases
“opening up” the capsid
 Uncoating of the genome initiates the eclipse period.

 Biosynthesis
 The eclipse period ends with the appearance of new
virions after virus assembly.
The virus eclipse period is defined as the interval between infection and the
first presence of new virus or virus components in cells.
BIOSYNTHESIS
◼genome synthesis
◼mRNA production
◼protein synthesis
Production of Viral Nucleic Acid and Protein
– Replication of DNA viruses in Nucleus
• exception Poxviruses in cytoplasma
– Replication of other RNA viruses in cytoplasma
• Exception: Retroviruses and Orthomyxoviruses in nucleus
• The replication strategies are different:

RNA viruses
• (+) stranded RNA acts a mRNA
• (-) stranded RNA makes a complementary (+) copies acting as mRNA
DNA viruses
• ‘early’ mRNA transcription from parental DNA
• ‘late’ mRNAs are transcribed from newly replicated DNA molecules
© 2012 Pearson Education, Inc.

Production of Viral Protein
• Viral Proteins
– Early proteins
– synthesized soon after infection
– necessary for replication of virus nucleic acid
– typically act catalytically
– synthesized in smaller amounts
– Late proteins
– Synthesized later
– Include proteins of virus coat
– structural components
– Synthesized in larger amounts
© 2012 Pearson Education, Inc.

DNA virus (HSV etc
replication.
1. Attachment
2. Penetration
3. Capsid dissolved
4. Biosynthesis
 Early mRNA synthesis
 Erarly protein synthesis
 Nucleic acid synthesis
 Late mRNA synthesis
 Late protein synthesis
5. Assebmly
6. Maturation and release
(-) stranded RNA
 Maturation : The stage of viral replication at which a
virus particle becomes infectious; nucleic acids and
capsids are assembled together.
 ASSEMBLY : All the structural components come together at in the
cell and the basic structure of the virus particle is formed.
 Assembly of naked DNA viruses in nucleus: Adenovirus, HPV, Parvovirus
 Assembly of enveloped DNA viruses in nucleus (HSV) or in
cytoplasma (Poxviruses, HBV..)
 Naked RNA viruses (eg: Picornavirus, Calicivirus) in cytoplasma
 Enveloped RNA viruses in cytoplasma (eg: Rubella, Rabies, HIV) or
nucleus (Influenza virus)
 RELEASE:
 Disintegration : naked virus cause the host cell lysis
 Budding: enveloped viruses
 Budding viruses do not necessarily kill the cell. Thus, some budding viruses may be
able to set up persistence
ASSEMBLY
 The site and mechanism of virion assembly in the cell depend on

 where genome replication occurs and
 whether the final structure is
 a naked capsid or
 an enveloped virus.
 Assembly of the DNA nucleocapsid for viruses other than poxviruses

occurs in the nucleus
 requires transport of the virion proteins into the nucleus.
 RNA virus and poxvirus assemblies occur in the cytoplasm.

RELEASE
 Naked capsid viruses are generally released after lysis of the cell.
 Release of most enveloped viruses occurs after budding from

the plasma membrane
Variation
There are two important variation which relate well with
medical practices
 Antigenicity variation: In most viruse the antigenicity is
stable but in some viruses such as influenza virus the
antigenicity may vary and cause the disease to epidemic.
 Influenza viruses have two types of changes:
 (1) antigenic shift,
 (2) antigenic drift
 Virulence variation (Virulent viruses): Less virulent
viruses always used in prevention.
Antigenic Shift
 This term denotes MAJOR changes in hemagglutinin

and neuraminidase resulting from reassortment of
gene segments involving two different influenza
viruses.
 When this occurs, worldwide epidemics may be the
consequence since the entire population is
susceptible to the virus.
Antigenic Drift
 This term denotes MINOR changes in hemagglutinin

and neuraminidase of influenza virus.
 This results from mutation in the RNA segments
coding for either the HA or NA
 This involves no change in serotype; there is merely
an alteration in amino acid sequence of HA or NA
leading to change in antigenicity.
Viroids
 Viroids: infectious RNA molecules that lack a protein coat
 Smallest known pathogens (246–399 bp)
 Cause a number of important plant diseases
 Small, circular, ssRNA molecules
 Do not encode proteins; completely dependent on host-encoded enzymes
 Viroids consist only of a short strand of circular RNA capable of self-
replication.
Prions (proteinaceous infectious particles)
 Prions: infectious proteins whose extracellular form contains no nucleic acid

 Host cell contains gene (PrnP) that encodes native form of prion
protein (PrPc) that is found in healthy animals
 PrPc is converted into the disease-causing form, which is PrPsc
 PrPsc may arise spontaneously in brain tissue, or
 it may originate from misfolded prions consumed in food that
eventually find their way into brain tissue.
 results in neurological symptoms of disease.
 These agents are associated with diseases such as Creutzfeldt-Jakob
disease in humans, scrapie in sheep & bovine, spongiform encephalopathy
(BSE) in cattle.
 Prions are extremely resistant to chemicals, heat, and radiation.
 There are no treatments for prion infection.
Prions
 A prion is a misfolded rogue form of a normal protein
(PrPc) found in the cell.
 This rogue prion protein (PrPsc),
 which may be caused by a genetic mutation or occur
spontaneously,
 can be infectious, stimulating other endogenous normal
proteins to become misfolded, forming plaques
Virusoids (Satellite RNAs)
 Non–self-replicating ssRNAs.
 Virusoids require the cell that also be infected with a
specific “helper” virus.
 Virusoids belong to a larger group of infectious
agents called satellite RNAs.
 Satellite RNAs may encode for proteins
 Satellite RNAs must coinfect with a helper virus to
replicate.
 One satellite RNA that has been described by some
scientists as a virusoid is the hepatitis delta virus
(HDV).
 The HDV helper virus is the hepatitis B virus (HBV).
 Coinfection with HBV and HDV results in more severe
pathological changes in the liver during infection
References
 Murray et al. Medical Microbiology, 2021
 Sherris Medical Microbiology 2022
82
ANTIBIOTIC
A substance produced by a micro-organism

that inhibits the growth of other
microorganisms (generally bacteria) at low
concentrations
Antibiotics either kill micro-organisms or

inhibit them,
▪ Bactericidal agents kill bacteria;
▪ bacteriostatic agents inhibit the growth of
bacteria.
2
CHEMOTHERAPEUTIC AGENT
Chemotherapeutic agent:
A chemical used to treat or cure disease.
A substance that interferes with the proliferation of cells (microorganisms and
other cells, e.g. cancer) at concentrations tolerable to the host (patient).
Antimicrobial agents:
chemical that can kills or inhibits growth of microorganisms
substances used in the treatment of infectious diseases, including antibiotics
and other antibacterials, antifungals, antiparasitics, and antivirals.
▪ anti-bacterial (bactericidal or bacteriostatic),

▪ anti-fungal (fungistatic or fungicidal),
▪ anti-viral, or
▪ anti-protozoal.
All antibiotics are classified in chemotherapeutic agents (used to treat bacterial

infections), but not all chemotherapeutic agents are antibiotics!
3
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With regard to organisms,
The CLSI defines as susceptible those isolates that are inhibited by typical
achievable concentrations of an antibiotic when the dosage recommended to
treat the infection site is used
The resistant category encompasses isolates that are not inhibited by the usual
achievable concentrations of an antibiotic agent or for which resistance mechanisms are
likely and clinical efficacy of antibiotics has not been reliably shown..
The intermediate category includes isolates that approach usually attainable

levels in blood and tissue by an antibiotic, and response rates are lower than
susceptible isolates
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Susceptible bacteria are inhibited at achievable nontoxic levels,
resistant strains are not
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•Bacteriostatic activity: Level of antimicrobial activity that inhibits the growth of an
organism.
•Bactericidal activity: Level of antimicrobial activity that kills the test organism.
•The MIC is the lowest concentration of antibiotic needed to inhibit the growth of an
organism
•The lowest concentration that kills 99.9% of the population is referred to as the
minimum bactericidal concentration (MBC).
7
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ANTIBIOTIC SPECTRUM OF ACTIVITY
Antibacterial spectrum: Range of activity of an antimicrobial against bacteria.
A broad-spectrum antibacterial drug can inhibit a variety of gram-positive and
gram-negative bacteria,
a narrow-spectrum drug is active against a limited variety of bacteria.
No antibiotic is effective against all

microbes
• Antibiotic combinations: Combinations of antibiotics that may be used
to
• (1) broaden the antibacterial spectrum for empirical therapy or the treatment of
polymicrobial infections,
• (2) prevent the emergence of resistant organisms during therapy,
• (3) achieve a synergistic killing effect.
• Antibiotic synergism:
• Combinations of two antibiotics have enhanced bactericidal activity
when compared with the activity of each antibiotic.
• Antibiotic antagonism:
• Combination of antibiotics in which the activity of one antibiotic
interferes with the activity of the other
▪ (e.g., the sum of the activity is less than the activity of the
most active individual drug).
• Additive effect:
• the potency of an antibiotic combination is roughly equal to the
combined potencies of each antibiotic singly
11
MPC: mutant prevention
concentration
THE SELECTIVE TOXICITY OF ANTIBIOTICS
means that antibiotics must be highly
effective against the microbe but
have minimal or no toxicity to
humans.
In practice, this is expressed by a

drug's therapeutic index (TI)
▪ the ratio of the toxic dose (to the patient) to the
therapeutic dose (to eliminate the infection).
▪ The larger the index, the safer is the

drug (antibiotic) for human use.
13
A CLINICALLY-USEFUL ANTIBIOTIC SHOULD HAVE
AS MANY OF THESE CHARACTERISTICS AS
POSSIBLE
➢ It should have a wide spectrum of activity

➢ It should be nontoxic to the host and without undesirable side effects.
➢ It should be nonallergenic to the host.
➢ It should not eliminate the normal flora of the host.
➢ It should be able to reach the part of the human body where the
infection is occurring.
➢ It should be inexpensive and easy to produce.
➢ It should be chemically-stable (have a long shelf-life).
➢ Microbial resistance shouldn’t be developed for it
14
BASIC MECHANISMS OF ANTIBIOTIC
ACTIVITY
❖Inhibition of cell wall synthesis

❖Inhibition of cell membrane function
❖Inhibition of protein synthesis
❖Inhibition of nucleic acid synthesis
❖Antimetabolites
15
Inhibition of cell wall
synthesis
16
Inhibition of cell wall synthesis
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INHIBITORS OF CELL WALL SYNTHESIS
-lactam antibiotics: Penicillins, cephalosporins, carbapenems,

monobactams…..
Glycopeptides:Vancomycin, Teicoplanin, Telavancin
Polypeptides:Bacitracin
Antibiotics effective against Mycobacteria: Isoniazid,

ethambutol, cycloserine and ethionamide
18
-LACTAM ANTIBIOTICS
They have a common -lactam ring structure.

▪ Penicillins (Penicillin G, ampicillin, amoxicillin ticarcillin ,piperacillin …)
▪ Cephalosporins (Cefazolin, Cephalexin, Ceftriaxone, Cefotaxime,Cefepime ..)
▪ Cephamycins (Cefoxitin, Cefotetan, Cefmetazole)
▪ Carbapenems (Imipenem, Meropenem, Doripenem, Ertapenem)
▪ Monobactams (Astreonam)
19
▪ -lactam/-lactamase inhibitors
▪ Selected penicillins such as ampicillin, amoxicillin, ticarcillin,
piperacillin have been combined with -lactamase inhibitors e.g
clavulonic acid, sulbactam, tazobactam, and avibactam.
▪ -lactamase inhibitors bind -lactamases and prevents enzymatic
inactivation of -lactam ring of antibiotics
• β-lactam antibiotic activity is enhanced in the presence of β-lactamase

inhibitors
• Clavulanate, sulbactam, and tazobactam are β-lactams that inhibit β-
lactamases via direct binding
• Avibactam inhibits β-lactamases via indirect means, and may provide superior
protection against highly drug-resistant bacteria
Penicillins
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Penicillin’s penetration of gram-negative outer membrane is often limited
A group of broader spectrum penicillins are able to traverse the outer
membrane of some gram-negative bacteria, and
some, such as the aminopenicillins ampicillin and amoxicillin, have excellent
activity against a range of gram-negative pathogens but not against
Pseudomonas aeruginosa
**the ureidopenicillin piperacillin, is active against Pseudomonas when

given in high dosage.
The cephalosporins are classified by generation
First generation cephalosporins cefazolin and cephalexin have a spectrum of

activity against gram-positive organisms.
In addition, they are active against some of the Enterobacteriaceae ,
Second-generation cephalosporins such as cefoxitin and cefaclor are resistant to

β-lactamases of some gram-negative organisms that inactivate first-
generation compounds.
They have expanded activity against Enterobacteriaceae species, ,
Third-generation cephalosporins, such as ceftriaxone, cefotaxime, and

ceftazidime, have an even wider spectrum;
they are active against gram-negative organisms
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The cephalosporins are classified by generation
Fourth-generation cephalosporins have enhanced ability to cross the outer

membrane of gram-negative bacteria as well as resistance to many gram-
negative β-lactamases.
Cefepime has activity against a wider spectrum of Enterobacteriaceae as well
as P. aeruginosa
Fifth-generation cephalosporins such as ceftolozane are built to kill highly drug-

resistant gram-negative bacteria, including P aeruginosa.
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Carbapenems.
• The carbapenems imipenem, meropenem, and doripenem have the broadest
spectrum of all β-lactam antibiotics.
• This fact appears to be due to the combination of easy penetration of gram-
negative and gram-positive bacterial cells and high level of resistance to β-
lactamases
Monobactams
• Monobactams (aztreonam) have poor affinity for the PBPs of gram-positive
organisms and strict anaerobes and thus demonstrate little activity against
them.
• They are highly resistant to hydrolysis by β-lactamases of gram-negative bacilli.
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• Cephamycins are a group of β-lactam antibiotics .
• Examples: Cefoxitin, Cefotetan, Cefmetazole
• They are sometimes classified as cephalosporins.

• Cephamycins are a very efficient antibiotic against anaerobic
microbes.
• In addition,
• Cephamycins have been shown to be stable against extended-
spectrum beta-lactamase (ESBL) producing organisms.
-LACTAM ANTIBIOTICS: ACTION
The backbone of the peptidoglycan consist of N-acetylglucosamin (NAGA) and N-acetylmuramic acid (NAMA)
connected by  1-4 linkages.
NAM and NAG chains (peptidoglycan layers) are cross linked with peptide bridges (pentaglysin bound) in the
peptidoglycan layer.
This structure is catalyzed by specific enzymes called penicillin binding proteins (PBPs),
PBPs are responsible for the final stages of bacterial cell wall assembly.
These enzymes are targets of β-lactam antibiotics.
Two of the PBP activities include DD-transpeptidase and DD-carboxypeptidase activities
-lactam antibiotics bind to specific PBPs in the bacterial cell wall and inhibits formation of
cross links between peptidoglycan layers.
-lactam antibiotics act as bactericidal agents on growing bacteria
27
-LACTAM ANTIBIOTICS: ACTION
-lactam antibiotics bind to specific PBPs in the bacterial cell wall and
inhibits formation of cross links between peptidoglycan layers.
-lactam antibiotics act as bactericidal agents on growing bacteria
12.03.19
Resistance to penicillin via modified cross-
linking enzyme.
29
RESISTANCE TO -LACTAM ANTIBIOTICS
1. The changes in the porin proteins alter the size or charge of these
channels,
▪ Beta lactams are exclude
▪ e.g.Gram negative bacteria, particularly Pseudomonas species
1. Overproduction of PBPs
1. Acquisition of a new PBP (methicillin resistance in Staphylococcus aureus)

2. Modification of existing PBP through recombination (e.g.penicillin
resistance in Streptococcus pneumoniae ) or a point mutation (e.g
penicillin resistance in Enterococcus faecium )
30
RESISTANCE TO -LACTAM ANTIBIOTICS-2
5-Production of -lactamases:
• Hydrolyse the antibiotic
• 200 different beta lactamases have been described
Some are specific for
▪ Penicillins (penicillinase),
▪ Cephalosporins (cephalosporinases )
▪ Carbepenems (carbepenemases)
▪ Some of them have a broad range activity,
▪ They can inactivate most -lactam antibiotics, (i.e.all penicillins and
cephalosporins
▪ These are called as extended-spectrum -lactamases (ESBLs)
▪ ESBLs are troublesome because they are encoded on plasmids that can be transferred
from organism to organism
31
GLYCOPEPTIDES
Vancomycin
• Distrupts peptidoglycan
synthesis in growing gram
positive bacteria
• Interacts with D alanine-D-
alanine termini of the
pentapeptide side chains,
which interferes the formation
of the cross-linkage between
the peptidoglycan chains
• In the sensitive bacteria,
cross-links cannot be formed
and the cell wall falls apart.
32
RESISTANCE TO VANCOMYCIN
Vancomycin is used for management of infections caused by oxacillin-
resistant staphylococci and other gram-positive bacteria resistant to -
lactam antibiotics
Vancomycin is inactive for gram (-) bacteria because its molecule is too large to
pass through the outer membrane.
Some organisms are intrinsically resistant to vancomycin (pentapeptide

terminates in D-alanine-D –lactate does not bind vancomycin)
Some Enterococcus species (E.gallinarum, E. casseliflavus) are resistant to

vancomycin intrinsically (they contain D-alanine-D-serine terminus)
▪ Some species of enterococci have acquired resistance on plasmids, this kind of

resistance is troublesome for treatment of enterococcal infections..
33
POLYPEPTIDES: BACITRACIN
➢ Bacitracin inhibits cell wall synthesis by interfering movement

peptidoglycan precursors through the cytoplasmic membrane
➢ Bacitracin is active for Staphylococci.
➢ Resistance to Bacitracin:
➢ The antibiotic cannot penetrate into the bacterial cell
34
ISONIASID, ETHIONAMIDE, ETHAMBUTOL,
CYLOSERINE
➢ They are used for mycobacterial infections

➢ They are all interfere the cell wall synthesis by
blocking mycolic acid / arabinogalactan
synthesis in the cell wall.
Resistance to these antibiotics

▪ reduced drug uptake into the bacterial cell
▪ alteration of the target sites.
35
Inhibition of cell membrane function
36
POLYMYXINS: Colistin (Polymyxin E), Polymyxin B
The polypeptide antimicrobial agents polymyxin B and colistin have a
cationic detergent-like effect.
They can be bactericidal for many gram-negative aerobic rods by

binding to lipopolysaccharides and the phospholipids in the outer
membrane.
They increased cell permeability and cell death.
37
Daptomycin binds irreversibly to the cytoplasmic membrane,
resulting in membrane depolarization and disruption of
the ionic gradients, leading to cell death.
It has potent activity against gram-positive bacteria, but

gram negative bacteria are resistant to daptomycin because
the drug cannot penetrate through the cell wall to the cytoplasmic membrane.
Daptomycin has good activity against multidrug-resistant

staphylococci, streptococci, and enterococci (including
vancomycin-resistant strains).
Inhibition of Protein
Synthesis
39
Inhibition of Protein Synthesis
12.03.19
Action of antimicrobials on protein synthesis.
Aminoglycosides (A) bind to multiple sites on both the 30S and 50S ribosomes in a
manner that prevents tRNA from forming initiation complexes.
Tetracyclines (T) act in a similar manner, binding only to the 30S ribosomes.
Chloramphenicol (C) blocks formation of the peptide bond between the amino acids.
Erythromycin (E) and macrolides block the translocation of tRNA from the acceptor to
the donor side on the ribosome.
12.03.19
Aminoglycoside
Action: The first aminoglycoside, streptomycin, binds to the 30S ribosomal
subunit, but the newer and more active aminoglycosides bind to
multiple sites on both 30S and 50S subunits.
This attachment to the ribosomes has two effects:

➢production of abnormal proteins as the result of misreading of the
messenger RNA (mRNA), and
➢interruption of protein synthesis by causing the premature release of the
ribosome from mRNA.
➢ Used to treat the infections caused by many gram (-) rods and some gram (+)
bacteria
kanamycin, amikasine, gentamicin, tobramicin are used commonly.
42
Aminoglycoside..
Actions:
Anaerobes are resistant to these antibiotics.
▪ Penetration of the aminoglycosides through the cytoplasmic membrane is
an aerobic energy dependent process ,
Streptococci and Enterococci are resistant to aminoglycosides,

➢because aminoglycosides cannot penetrate to the cell wall of these bacteria
.
➢Treatment of these bacteria infections requires coadministration of an
aminoglycoside with an cell wall inhibitor (e.g. penicillin, ampicillin,
vancomycin)
43
• Eukaryotic ribosomes are resistant to aminoglycosides, and the
antimicrobials are not actively transported into eukaryotic cells.
• These properties account for their selective toxicity and also

explain their ineffectiveness against intracellular bacteria
such as Rickettsia and Chlamydia.
12.03.19
Resistance to Aminoglycosides
1. Mutations of the ribosomal binding site

2. Enzymatic modification of the antibiotic (the most
common
This is accomplished by the action of phosphotransferases, adenyltransferases,
and acetyltransferases on the amino and hydroxyl groups of the antibiotic.
3. Decreased uptake of the antibiotic into the bacterial
cell
4. increased expulsion of antibiotic from the cell
45
Inhibition of Protein Synthesis:
Tetracyline, Doxycycline, Minocycline
Action:
▪ inhibit the protein synthesis by binding 30s ribosomal subunit,
(blocking the binding aminoacyl-tRNA to the 30s ribosome + mRNA complex)
Their effect is reversible.

They are bacteriostatic
Effective in the treatment of infections caused Rickettsia, Chlamydia and
Mycoplasma.
Resistance:
▪ Decreased penetration of the antibiotic into the bacterial cell.
▪ Active efflux of the antibiotic out of the cell (most common)
▪ Alteration of the ribosomal binding site
▪ Enzymatic modification of the antibiotic
▪ The production of proteins similar to elongation factors.
46
Inhibition of Protein Synthesis: Oxazolidinones
➢ Blocks inition of the protein synthesis at 50s ribosome

➢ They stop translation of messenger RNA (mRNA) into proteins in the
ribosome
➢ This is a unique mechanism and cross resistance doesn’t occured
with other protein inhibitors
➢ Linezolid is the agent currently used.
➢ Linezolid has activity against all staphylococci, streptococci and
enteroccoci
➢ Activity against gram-positive bacteria resistant to other agents
47
Inhibition of Protein Synthesis:
Chloramphenicol
• It has bacteriostatic effect,
• binds reversibly to 50s ribosomal subunit thus blocks peptide
elongation (blocking the action of peptidyl transferase),
• chloramphenicol is a broad-spectrum antibiotic with a wide range of
activity against both aerobic and anaerobic species
It is limited used
• it disrupts protein synthesis in human bone marrow cells (1/24000
treated patients)
Resistance:
• Producing plasmid encoded enzyme that catalyse acetylation of
the chloramphenicole,
48
Inhibition of Protein Synthesis: Macrolides
The macrolides erythromycin, azithromycin, and clarithromycin affect
protein synthesis at the ribosomal level by binding to the 50S subunit
and blocking the translocation reaction
• Blocks protein elongation
• Bacteriostatic activity
• They have been used
▪ To treat pulmonary infections caused by Mycoplasma, Chlamydia,
Legionella
▪ To treat infections caused by Campylobacter and gram (+) bacteria in
patients allergic to penicillin.
Resistance to macrolides
▪Methylation of the 23s ribosomal RNA, prevents binding by the antibiotic
▪ Enzyme production inactivates the macrolides
▪
49
Inhibition Of Protein Synthesis: Clindamycin
Blocks protein elongation by binding 50s ribosome

Clindamycin is active against staphylococci and anaerobic gm(-)
bacteria
Resistance : methylation of the 23S rRNA
Both erithromycin and clindamycin can induce enzymatic resistance
(plasmid mediated)
Cross resistance is observed between these two classes of antibiotic
50
Inhibition Of Protein Synthesis: Streptogramins
Group A and Group B streptogramins act synergistically to inhibit protein

synthesis
Quinupristin-dalfopristin is currently available (synercid)
Dalfopristin prevents peptid chain elongation,

Quinupristin initiate premature release of peptide chain
This drug is used primarly Vancomycin resistant Enterococcus

faecium infections
Also it is active Staphylococci, Streptococci
51
Inhibition of nucleic acid
synthesis
52
Inhibition of Nucleic acid synthesis
12.03.19
Antimicrobials acting on nucleic acids
• Sulfonamides block the folate

precursors of DNA synthesis,
• metronidazole inflicts breaks in the
DNA itself,
• rifampin inhibits the synthesis of
RNA from DNA by inhibiting RNA
polymerase, and
• quinolones inhibit DNA
topoisomerase and thus prevent the
supercoiling required for the DNA to
“fit” inside the bacterial cell.
12.03.19
Inhibition of Nucleic Acid Synthesis
Quinolones
• Nalidixic acid, Ciprofloxacin, Levofloxacin, Gatifloxacin, Moxifloxacin
• The target of the quinolones are DNA gyrase and topoisomerase IV, the
enzymes responsible for nicking, supercoiling, and sealing bacterial DNA during replication .
• Quinolones are active against both Gram (+) and Gram (-) bacteria
• The fluoroquinolones are highly active and bactericidal against a wide

range of aerobes and facultative anaerobes.
• However, strict anaerobes are generally resistant.
• Resistance:
▪ chromosomal mutation in the structural genes for topoisomerases
▪ Decreased drug uptake
55
ACTION OF QUINOLONES
56
Inhibition Of Nucleic Acid Synthesis
Rifampin And Rifabutin
They inhibit the initiation of RNA synthesis by binding to DNA

dependent RNA polymerase
Rifampin is bactericidal for Mycobacterium tuberculosis,
Rifabutin is active against Mycobacterium avium
Resistance:
▪ Mutation in the chromosomal gene (Gram + bacteria)
▪ Gram (-) bacteria are resistant intrinsically to rifampin (result of
decreased uptake of the hyrophobic antibiotic)
57
Inhibition Of Nucleic Acid Synthesis
Metronidazole
• Distrupts bacterial DNA
• The antimicrobial properties of metranidazole stem from the reduction of its nitro group
under anaerobic conditions by bacterial nitroreductase,
• The reduction products act on the cell at multiple points; the most lethal of these effects is
induction of breaks in DNA strands.
• Effective Trichomonas vaginitis, amebiasis, giardiasis and anaerobic

infections.
• Resistance :
▪ Decreased uptake of the antibiotic
▪ Elimination of the cytotoxic compounds before they can interact host DNA.
58
Folate Inhibitors
• They interfere with the synthesis of folic acid by bacteria
• They have selective toxicity because mammalian cells use preformed
folate from dietary sources.
• Folic acid is derived from para-aminobenzoic acid (PABA), glutamate, and a
pteridine unit.
• Folic acid is indirectly essential for the synthesis of nucleic acids and
proteins
• The major inhibitors of the folate pathway are
• the sulfonamides ,
• trimethoprim,
• para-aminosalicylic acid,
• the sulfones.
• ….
12.03.19
Antimetabolites
Sulfonamides and trimethoprim interfere with folic
acid metabolism at two step
This inhibition blocks the formation of thymidine,
some purines, methionin and glycine.
Trimethoprim is commonly combined with

sulfamethoxazole for synergistic effect.
Trimethoprim sulfamethoxazole is used
particularly for acut and chronic urinary tract
infections, and the lower respiratory tract infections
otitis media and uncomplicated gonorrhoae.
Also is effective Pneumocystis jiroveci infections
Sulfonamides are effective for

Gram (+) and Gram(-) bacteria,
Chlamydia,
Nocardia and some protozoa.
60
Antimetabolites
Dapsone and p-aminosalicylic acid are also antifolates

they are useful for mycobacterial infections.
Resistance:
▪ permeability barriers (e.g.pseudomonas)
▪ Decreased affinity to target enzyme
▪ Bacteria can use exogenous thymidine (Enterococci)
61
Overview of Mechanisms of Action of Antimicrobial
Drugs
62
63
Overview of Mechanisms of Antimicrobial
Resistance
1) Altering the target protein (e.g., change in penicillin-binding protein 2b in
pneumococci, which results in penicillin resistance),
(2) Upregulating the production of enzymes that inactivate the antimicrobial

agent (e.g., Staphylococci resistant to penicillin G produce a beta-lactamase
that destroys drug)
(3) Microorganisms change their permeability to the drug; downregulating or

altering an outer membrane protein channel that the drug requires for cell
entry (e.g., OmpF in E coli),
(4) Upregulating pumps that expel the drug from the cell (efflux of
fluoroquinolones in S aureus)
64
Overview Of Mechanisms Of Antimicrobial Resistance-2
(5) Microorganisms develop an altered metabolic pathway that by

passes the reaction inhibited by the drug.
Example: Some sulfonamide-resistant bacteria do not require
extracellular PABA (p-aminobenzoic acid)
6) Microorganisms develop an altered enzyme that can still

perform its metabolic funtion
7) Genetic resistance: For any antimicrobial, there are bacterial
species that are typically within its spectrum and those which
are not. The resistance of the latter group is referred to as
intrinsic or chromosomal to reflect its inherent nature.
65
Antimicrobial resistance mechanisms. A. Exclusion barrier. B.
Altered target. C. Enzymatic inactivation.
12.03.19
Antiviral Agents and Infection Control
It is more difficult to inhibit viral replication without also being

toxic to the host.
Most antiviral drugs are targeted toward viral-encoded enzymes

or structures of the virus that are important for replication.
Most of these compounds are classic biochemical inhibitors of

viral-encoded enzymes.
Some antiviral drugs are actually stimulators of host innate

immune protective responses
67
SELECTED ANTIVIRAL AGENTS
• Inhibitors of Attachment :
• Attachment to a cell receptor is a virus-specific event.
• Antibodies can bind to the extracellular virus and prevent this attachment.
• Inhibitors of Cell Penetration and Uncoating :
• Amantadine and rimantadine inhibit viral uncoating
• Neuraminidase Inhibitors:
• Oseltamivir and zanamivir are antiviral agents that inhibit the
neuraminidase of influenza A and B viruses
• Nucleotide Analogs:
• Cidofovir inhibits viral DNA polymerase
• Ribavirin is another analog of the nucleoside guanosine.
12.03.19
SELECTED ANTIVIRAL AGENTS..
Inhibitors of Nucleic Acid Synthesis:
• ✺Idoxuridine and trifluorothymidine block DNA synthesis
• ✺Acyclovir is effective against the herpesviruses, which induce thymidine kinase
• ✺Acyclovir inhibits viral DNA polymerase and terminates viral DNA chain growth
Interferons are host cell-encoded proteins synthesized in response to

double-stranded RNA (dsRNA) that circulate to protect uninfected cells by
inhibiting viral protein synthesis.
Interferon α is beneficial in the treatment of chronic active hepatitis B
and C infection.
12.03.19
70
General scheme of antiviral action
12.03.19
Sites of action of antifungals
Caspofungin
Micafungin
Anidulafungin
amphtericin B 72
LABORATORY DIAGNOSIS OF
INFECTIOUS DISEASES
Prof Dr Rıza DURMAZ
2022
Microbiological investigations
The Proper Diagnosis of an Infectious
Diseases Requires the followings
• Taking complete patient history
• Conducting an exact physical examination of the

patient
• Carefully evaluating the patient’s signs and symptoms
• Proper selection, collection, transport and

processing of appropriate clinical specimens
Proper Selection & Collection of Clinical
Specimen
• The specimen must be properly selected
– Blood in culture bottle, not clotted blood
– Sputum, not saliva
– Pus, not swab…
• The specimen must be properly and carefully
collected
– Take an mid-stream urine
• Avaids contamination with perineal flora
– CSF
• Avoid contamnation
– Blood cultures
• Avaid contamination with skin organisms
Proper Collection of Clinical Specimen
• The specimens should be collected from the site
– where the suspected pathogen is most likely to be found and
– where the least contamination is likely to occur.
• Whenever possible, specimens should be obtained before

antimicrobial therapy has begun
• If possible, collect the specimen in the acute phase of the

• Infection
• A sufficient quality of the specimen must be obtained to

provide enough material for all required diagnostic tests.
• Throat culture for GAS is the diagnostic
gold standard and has a sensitivity of 90%
to 95%.
• In collecting pharyngeal specimens for
streptococcal cultures, it is important to
vigorously swab both of the tonsillar
areas as well as the posterior pharynx.
• Specific efforts should be made to swab

involved areas directly.
• The tongue and other oral structures should

be avoided with the swab to minimize
contamination with oral biota and dilution of
Specimen collection from the
the specimen.
oropharynx
Proper Collection of Clinical Specimen
• Collect the specimen using the proper
technique
• Package the specimen in a container or transport
medium designed to maintain the viability of
the organisms and avoid hazards that result from
leakage.
• Label the specimen
• Transport the specimen to the laboratory

promptly
• Notify the laboratory in advance if unusual

pathogens or agents of bioterrorism are suspected.
Case investigation form
Patient information
– age (or date of birth), sex, complete address
Clinical information
– date of onset of symptoms,
– clinical and immunization history,
– risk factors or contact history where relevant,
– anti-microbial drugs taken prior to specimen collection
Laboratory information
– acute or convalescent specimen
– other specimens from the same patient
Line listing – if large number of patients
Collection Procedures
• Specimens for microbiology cultures should be collected in
sterile containers
– except for stool specimens, which can be collected in clean, leakproof
containers.
• Generally, swabs are not recommended for collection
because
– they do not provide sufficient quantity,
– They are easily contaminated, and
– They can become dried out, leading to a loss of
organisms.
• Swabs are appropriate for specimens from the upper
respiratory tract, external ear, eye, and genital tract.
• Swab collection systems are available that provide

transport media and protect the specimen from drying.
SWAB
• The tips of swabs may contain cotton, Dacron, rayon, or calcium alginate.
• Cotton-tipped swabs tend to have excessive fatty acids that may
be toxic to certain bacteria.
• Dacron or rayon polyester swabs have a wide range of uses.
Collection Procedures
• A wound is not an appropriate specimen source
when the exact anatomic site is not provided.
– Before the specimen is collected, the area
should be cleansed to eliminate as much of
the commensal flora as possible.
• The specimen should be collected by needle
aspiration whenever possible, rather than by
swab from the advancing margin of the lesion.
– Aspirated material should be placed into a

sterile tube or transport vial
Specimen Storage
• Some specimens that will not be transported or processed immediately can
be maintained by being stored under certain conditions.
• Some specimens, such as urine, stool, sputum, bronchial secretions, swabs

(not for anaerobes), foreign devices such as catheters, and viral specimens,
– can be maintained at refrigerator temperature (4° C) for 24 hours.
• Pathogens that are cold sensitive may be found in other specimens, and
those specimens should be kept at room temperature if culture is to be
performed.
– This includes samples that might contain anaerobic bacteria as well as
most other sterile body fluids, genital specimens, and ear and eye
swabs
Specimen Storage
Before sending specimens
DIAGNOSTIC TESTS:
A-Direct tests B-Indirect tests
• Direct: Demonstration of the presence of
an infectious agent
– Culture
– Microscopy
– Macroscopic observation
– Molecular methods such as PCR
• Indirect: Demonstration of presence of
antibodies to a particular infectious agent
– Serology
Laboratory Investigation of Microbial infections
1-Macroscobic observation
2-Microscopy
3- Culture techniques
4- Animal pathogenicity test
5- Serological tests
6- Molecular biology techniques

Macroscopic observation
• if gas, foul smell, or

sulfur granules are
present
– Anaerobic cultures may
be indicated
Adult forms of parasites in stool
Ascaris
Microscopy
• Microscopy can be done quickly, but accuracy depends on

the experience of the microscopist and quality of
equipment
• Microscopy provide very useful data about

– motility,
– morphology, and
– staining characterisitcs of the microorganisms
Microscopy provide very useful data
• It can be used to determine the quality of the

specimen.
– Sputum specimens that represent saliva
rather than lower respiratory tract secretions
can be determined by the quantitation of
WBCs or epithelial cells
• Absence of WBCs may indicate that the
sample may not have been taken from
the actual site of infection.
• It can give an indication of the infectious

process involved.
– Gram stain of a sputum specimen revealing
WBCs and gram-positive diplococci is
indicative of Streptococcus pneumoniae
Microscopy provide very useful data
• The routine culture workup can be guided by the

results of the smear.
• It can dictate the need for nonroutine or additional

testing.
– The presence of fungal elements notify the
physician to request a fungus culture
• Microscopic examination can be done on
– unstained samples= wet mount examination
• fungi, parasites (including helminth eggs and
larvae), vaginal clue cells, motile organisms (eg,
Trichomonas), and syphilis (via darkfield
microscopy).
– stained samples
• Gram stain, acid-fast stains,……
Trichomonas vaginalis
Syphilis in dark-field microscop

Stained samples:
• Microscopic examination of a stained smear is usually the first
step in detecting and identifying disease.
• It provides important clues regarding the organism's size,
shape and structural composition
Simple stain: methylene blue stain
Gram stain: differentiation between Gm+ve and Gm–ve bacteria

. Primary stain (Crystal violet)
. Mordant (Grams Iodine mixture)
. Decolorization (ethyl alcohol)
. Secondary stain ( Saffranin)
Acid-fast stain: staining acid fast bacilli

. Apply strong carbol fuchsin with heat
. Decolorization (Acid and ethyl alcohol
. Counter stain (methylen blue)
Microscopy-Stained preparations
Simple stain: Staining can be

performed with basic dyes such as
crystal violet or methylene blue,
positively charged dyes
Display size and shape of

microorganisms
• Gram stain:
• The Gram stain classifies bacteria two main groups:
– Gram-positive—blue color
– Gram-negative—red color
• It displays cell morphology (eg, bacilli, cocci) and cell
arrangement (eg, clumps, chains, diploids).
• These characteristics can direct antibiotic therapy pending
definitive identification
Gram stain of CSF — N. meningitidis: intra-cellular, Bacillus anthracis: Gram positive basilli and
Gram-negative diplococci. PNL
Gram staining
Gram-stained smear of sputum acceptable for

culture, with white blood cells and gram-positive
diplococci.
Gram staining
Abscess aspirate, smear, Gram stain. Amniotic fluid.Gram-positive bacilli, small.

Purulence, heavy. Gram-positive cocci, groups, Morphology consistent with Listeria
extracellular. Impression: staphylococcal monocytogenes. Impression: congenital
disease listeriosis
• Acid-fast stains:
• These stains are used to identify acid-fast organisms
(Mycobacterium sp) and moderately acid-fast organisms
(primarily Nocardia sp).
• These stains are also useful for staining Rhodococcus and
oocysts of some parasites (eg, Cryptosporidium).
Cryptosporidium in stool
sample
Mycobacterium sp in clinical
specimens-
• Fluorescent stains:
• These stains allow detection at lower concentrations
(1 × 104 cells/mL).
– Examples are acridine orange (bacteria and fungi),
auramine-rhodamine and auramine O (mycobacteria), and
calcofluor white (fungi, especially dermatophytes).
Expectorated sputum, concentrated

smear, fluorochrome
acid-fast stain, fluorescent
microscopy.
Impression: mycobacterial disease.
2- Culture Techniques:
Gold standart for laboratory diagnosis of many infectious
disease
* Culture media are used for:
- Isolation and identification of pathogenic organisms
- Antimicrobial sensitivity tests
* Types of culture media:
a. Liquid media:
- Growth of bacteria detected by turbidity
b- Solid media:
- Colonial appearance
- Hemolytic activity
- Pigment production
Types of culture media
• Nonselective media support the growth of most
nonfastidious microbes
• Selective media support the growth of one type or group

of microbes but not another
• Differential media allow grouping of microbes based on

different characteristics demonstrated on the medium.
• Enriched media contain growth enhancers that are added

to nonselective agar to allow fastidious organisms to flourish
Culture Media Selection
• Based on the type of specimen submitted for culture and
the organisms likely to be involved in the infectious
process, proper culture medium or media is selected.
• Specimens in which fastidious pathogens are more likely

involved require enrichment medium.
– CSF---Chocolate agar
• Specimens that are collected from a site containing normal
biota require differential and or selective medium
– Stool sample---MacConcay agar, Endo Agar..
3- Animal pathogenicity
* Animal pathogenicity test:
Animals commonly used are guinea pigs, rabbits, mice
* Importance of pathogenicity test:

- Differentiate pathogenic and non pathogenic
- Isolation organism in pure form
- To test ability of toxin production
- Evaluation of vaccines and antibiotics

4-SEROLOGIC DIAGNOSIS
• Immunologic techniques are used
– To detect, identify and quantitate antigen
in clinical samples/ Direct serologic test
• Detect Streptococcus pneumoniae, N.
meningitidis, and/or Haemophilus influenzae
capsular antigens in CSF
– To evaluate the antibody response to
infection /Indirect serologic tests
Antijen detection
• Microbial antigen detection provides direct

evidence of infection, and is preferred for
diagnosis of infection over antibody detection
(indirect evidence of infection)
• However, not all infectious agents have

available antigen assays
Serological identification
A- Direct serological tests:
- Identification of unknown organism
- Detection of microbial antigens by using specific
known antibodies
- Serogrouping and serotyping of isolated organism
B- Indirect serological tests:

- Detection of specific and non specific antibodies
(IgM & IgG) by using antigens or organisms
• METHODS OF DETECTION
OF ANTIBODIES
1. Immuno-precipitation
Assays
2. Complement Fixation
3. Neutralization
4. Agglutination
5. Immunofluorescence
6. Enzyme Immunoassay
7. Radioimmunoassay
The details of these tests will be learned in the 2nd

grade in immunology
Some of the different diseases that can
be detected by serologic tests
• Syphilis
• Tularemia
• Brucellosis
• Human immunodeficiency virus (HIV)
• Viral hepatitis (HAV, HBV, HCV, HDV, HEV)
• Measles
• Rubella
• Influenza
Molecular diagnosis
• Genomic approaches are becoming the new gold standard for
specific types of microorganisms and are continuing to expand in
applications
• Molecular methods are particularly useful for agents that are difficult to
culture , no available culture system, or take a long time to grow
on culture media.
• Several nucleic acid detection technologies are
in use including:
• Probe hybridization (PH),
• Polimerase chain reaction (PCR),
• Transcription based/mediated amplification
(TMA),
• Ligase chain reaction (LCR),
• Strand displacement amplification (SDA)
• The Qβ replicase system.
Applications in microbiology
1. Classification of organism based on genetic relatedness

(genotyping)
2. Identification and confirmation of isolate obtained from culture
3. Early detection of pathogens in clinical specimen
4. Rapid detection of antibiotic resistance
5. Detection of mutations
6. Differentiation of toxigenic from non-toxigenic strains
7.Detection of microorganisms that lose viability during
transport, impossible, dangerous and costly to culture, grow
slowly or present in extremely small numbers in clinical specimen
Some organisms detected by
PCR
M. tuberculosis HIV Adeno
Legionellae HTLV Rabies
B. burgdorferii CMV Parvo B19
H. influenzae HSV Arbo
B.pertussis HHV Yellow Fever
N. meningitidis
VZV Lassa
T. pallidum
EBV JC/BK
H. pylori
F. tularensis Hepatides
C. difficile HPV Candidae
E. coli Rubella Cryptococcus
T. whipelii Influenza Trypanosoma
van, mec Rhino Toxoplasma
Env Naegleria….
Kaynaklar
• http://www.authorstream.com/Presentation/aSG
uest116180-1211757-13-diagnosing-infectious-
diseases/
• Murray PR, Rosenthal KS, Pfaller MA Medical
Microbiology, Elsevier Mosby.2020
• Jawetz, Melnick  Adelberg’s Medical
Microbiology 2010.
• Mahon C et al. Textbook of Diagnostic
Microbiology, Sixth Edition 2019
Introduction to Parasitology
What is a parasite?
• Parasitism is a type of symbiotic
relationship between two different
organisms.
• The parasite benefits from a prolonged,
close association with the host, which is
harmed
Medical parasitology
– Parasitic infections must be of concern to all health care workers
• Increasingly tourists, missionaries, Peace Corps volunteers, and others are visiting and working for
extended periods of time in exotic, remote parts of the world
– Approximately 30 % of the world's population is infected with the nematode

Ascaris lumbricoides
CLASSIFICATION AND STRUCTURE
• The parasites of humans are classified within the four eukaryotic
kingdoms: Protozoa, Animalia (Metazoa), Fungi,Stramenopila (formerly
Chromista)
• The Protozoa and Stramenopila are
single celled-organisms.
• Microsporidians are also

single-celled organisms and were
previously classified among the
protozoans; however,
– they now have been reclassified with the
Fungi.
• The members of the kingdom

Animalia, also known as metazoans,
are multicellular animals
Diversity among Protozoa
Plasmodium
Sporozoites 1 Infected mosquito bites 2 Sporozoites
in salivary human; sporozoites undergo
gland migrate through schizogony in
bloodstream to liver cell;
liver of human merozoites are
produced
9 Resulting sporozoites
migrate to salivary glands
of mosquito
3 Merozoites
Sexual released into
reproduction bloodsteam from
liver may infect
Asexual new red blood
8 In mosquito’s cells
Zygote digestive tract, reproduction
gametocytes unite
to form zygote Intermediate host
Female
gametocyte
4 Merozoite develops
Male into ring stage in red
gametocyte blood cell
Ring
stage
5 Ring stage
grows and
Definitive host divides,
producing
7 Another mosquito bites 6 Merozoites are released merozoites
infected human and ingests when red blood cell
gametocytes ruptures; some merozoites
infect new red blood cells,
and some develop into male
and female gametocytes
Merozoites
Figure 12.19
Giardia
Plasmodium
Classification
• Traditionally, parasite classification has taken into account
– morphology of intracytoplasmic structures, such as the nucleus,
– the type of locomotive organelles,
– the mode of reproduction
The new taxonomic consensus has emerged based mainly on the
biochemistry and molecular biology of lower organisms
(e.g., Protozoa, Fungi, and Stramenopila).
Comparisons of small subunit ribosomal ribonucleic acid (SSU
rRNA) and protein sequences
Factors Associated with Parasite Pathogenicity
• Infective dose and exposure

• Penetration of anatomic barriers
• Attachment
• Replication
• Cell and tissue damage
• Disruption, evasion, and inactivation of host defenses
Exposure and Entry
• Parasites are virtually always acquired from an exogenous source.
• Inoculum size and duration of exposure greatly influence the

disease-causing potential of an organism.
– The severity of illness caused by many parasites is related to the infecting dose and
the number of organisms.
• Likewise, the route of exposure is critical for most organisms.

– For example, Entamoeba histolytica are unlikely to cause disease on exposure to
intact skin but may cause severe dysentery after oral ingestion.
Transmission of parasitic diseases
• Transmission of parasitic diseases is frequently facilitated by
environmental contamination with human and animal wastes.
– Fecal-oral route is the most common route:For examples
• Entamoeba histolytica
• Giardia lamblia
• Enterobius vermicularis
• Ascaris lumbricoides
– Many parasitic diseases are acquired via the bites of arthropod vectors.
For examples
• African trypanosomiasis
• Babesiosis
• Chagas disease
• Leishmaniasis
• Malaria
– Blood transfusion or by sharing needles or syringes contaminated with blood
For examples
• Babesiosis
• Chagas disease
• Leishmaniasis
• Malaria
• Toxoplasmosis
– Venereal route:Trichomonas vaginalis
Transmission of parasitic diseases-
Blood
• Many bloodborne parasites are spread
by insects (vectors), so they are also
referred to as vectorborne diseases.
• Babesiosis
• Chagas disease
• Leishmaniasis
• Malaria
Food
• Parasites can be transmitted by food
including many protozoa and helminths eggs.
• Undercooked fish,meat, raw aquatic plants
such as watercress, raw vegetables
• Contaminated by human or animal feces
• Some foods are contamined by food service
workers
Entamoeba histolytica
Cryptosporidium
Giardia intestinalis,
Cyclospora cayetanensis
Toxoplasma gondii,
Trichinella spiralis
Taenia spp.
Water
• Parasites can live in natural water
sources.
• When water becomes contaminated by
parasites, it can cause a variety of
illnesses, even death.
• Schistosomiasis
• Amebiasis
• Cryptosporidiosis
• Giardiasis.
Factors Associated with Parasite Pathogenicity
• Infective dose and exposure:

• Penetration of anatomic barriers
• Attachment
• Replication
• Cell and tissue damage
• Disruption, evasion, and inactivation of host defenses
Adherence and Replication
• Most infections are initiated by the
attachment of the organism to host tissues
• Followed by replication to colonization.
• Specific surface structures:
– Surface glycoproteins
– Fibronectin
– N-acetylglucosamine conjugates.
Examples of Parasitic Adherence Mechanisms
Organism Disease Target Mechanism of Attachment and Receptor
Plasmodium vivax Malaria Red blood cell Merozoite (noncomplement-mediated
attachment), Duffy antigen
Plasmodium falciparum Malaria Red blood cell Merozoite and glycophorin A and B
Babesia species Babesiosis Red blood cell Complement-mediated C3b receptor
Giardia lamblia Diarrhea Duodenal and jejunal Trypsin-activated G. lamblia lectin and
epithelium mannose-6-phosphate.
G. lamblia adherence molecule-1 on disk
Entamoeba histolytica Dysentery Colonic epithelium Lectin and N-acetylglucosamine

conjugates
Trypanosoma cruzi Chagas' disease Fibroblast Penetrin and fibronectin receptor
Leishmania major Leishmaniasis Macrophage C3bi and CR3

Leishmania mexicana Leishmaniasis Macrophage Surface glycoprotein (gp63) and CR2
Necator americanus Hookworm Intestinal epithelium Mechanical and biting mouthparts

Ancylostoma duodenale
Replication
• Most protozoan parasites replicate intracellularly

or extracellularly in the human host.
• Temperature may also play an important role in

the ability of parasites to infect a host and cause
disease.
– Leishmania donovani replicates well at 37° C and causes

visceral leishmaniasis
• In contrast,
– L. tropica grows well at 25° C to 30°C but poorly at 37° C
and causes an infection of the skin without involvement of
deeper organs.
Cell and Tissue Damage
• Parasitic protozoa and helminths

are generally not known to
produce toxins
• However, parasitic disease can

be established by
– toxic products,
– mechanical tissue damage,
– immunopathologic reactions.
Cell and Tissue Damage…
TOXİC PRODUCTS
• Proteases and phospholipases may be

secreted and are released on the
destruction of the parasites.
• These enzymes can cause

– host cell destruction,
– inflammatory responses, and
– gross tissue pathology.
• E. histolytica produces proteases and

phospholipases that can degrade
epithelial cell layers.
BLOCKAGE
• Large adult Ascaris organisms can
physically block the intestine and
the bile ducts.
• Likewise, blockage of lymph flow,
leading to elephantiasis, is
associated with the adult Wuchereria
organisms
HYPERSENSİTİVİTY
REACTİONS
• Migration of helminths
(usually larval forms) through
body tissues such as the skin,
lungs, liver, intestines, eyes,
and CNS can damage the
tissues directly and initiate
hypersensitivity reactions.
During a parasitic infection,
• Immunopathologic reactions range from acute anaphylactic reactions to
cell-mediated delayed hypersensitivity reactions.
• Many inflammatory changes become irreversible, producing functional
changes in tissues.
Diagnosis in parasitic infection
• Diagnosis of parasitic infections requires laboratory support,
– since the signs and symptoms are often nonspecific.
• A variety of methods and specimens are used for diagnosis.
• Since the most common parasites are enteric

– Microscopic examination of fecal specimens is done more often than any
other laboratory procedure.
– Culturing has little application in the diagnosis of most parasitic infections

• For example, Trichomonas vaginalis and Entamoeba histolytica infections.
– Immunodiagnostic tests are useful in several infections

• including extraintestinal amebiasis, visceral larva migrans, and trichinosis.
Laboratory Methods for Diagnosing
Parasitic Disease
• Macroscopic examination
• Microscopic examination
– Wet mount
– Permanent stains
– Stool concentrates
– Serologic examination
– Antibody response
– Antigen detection
• Nucleic acid hybridization
– Probes and amplification techniques
– Detection
– Identification
• Culture
• Animal inoculation
• Xenodiagnosis
Fecal Specimen Collection
• Fecal specimens should be collected in
clean, wide-mouthed, waterproof containers
with a tight-fitting lid
• For a routine parasitic examination, a

total of 3 fecal specimens is
recommended.
• The series of three specimens should be

collected within no more than 10 days
Fecal Specimen Collection
• Unpreserved formed fecal specimens should arrive in the
laboratory within 2 hours after passage.
• If the stool is liquid and thus more likely to contain trophozoites, it

should reach the laboratory for examination within 30 minutes.
• If examination is not possible within the recommended time

limits, all fresh fecal samples should be placed into
preservatives
– such as 10% formalin, polyvinyl alcohol (PVA), merthiolate-iodine-formalin
(MIF), or sodium acetate formalin (SAF).
Macroscopic Examination
• The fecal specimen
should be examined
for the presence of..
- Blood
- Mucus
- Worms
- Proglottids
Direct Wet Mount
• Fresh stools should be
examined under the
microscope using the
saline and iodine
wet-mount technique to
detect (material from sputum,
urine, vaginal swabs, duodenal aspirates, sigmoidoscopy,
)
abscesses, and tissue biopsies
- Motile trophozoites
- Larvae
- Helminth eggs
- Protozoan cysts
- Host cells
such as leukocytes and red blood cells
Permanently Stained Slides
• The common permanent stains used are
– trichrome, iron hematoxylin, and phosphotungstic
acid-hematoxylin.
Collection and Examination of Specimens Other Than Stool
Perianal Specimens
• The collection of perianal specimens is
frequently necessary to diagnose pinworm
(E. vermicularis) and occasionally Taenia
(tapeworm) infections.
• The methods include the preparation of a clear cellulose

tape slide or an anal swab.
• The tape method requires that the adhesive surface of the

tape be pressed firmly against the right and left perianal
folds and then spread onto the surface of a microscope
slide.
• The slides or swabs should be kept at 4° C if transport to

the laboratory is to be delayed.
Collection and Examination of
Specimens Other Than Stool
Sputum
• Occasionally, intestinal parasites may be
detected in sputum.
• These organisms include

– the larvae of Ascaris, Strongyloides, and
– intestinal protozoa such as E. histolytica and
Cryptosporidium species.
• Microscopic examination
– saline wet-mount
– permanent stain preparations.
Collection and Examination of Specimens Other Than Stool
Urine
• Examination of urine specimens

may be useful in diagnosing
infections caused by
Schistosoma haematobium and
Trichomonas vaginalis.
Urogenital Specimens
• Urogenital specimens are

collected if infection with T.
vaginalis is suspected.
• Identification is based on
wet-mount preparation
examinations of vaginal and
urethral discharges, prostatic
secretions, or urine sediment.
Parasitic Infections of Blood and
Tissue
prepared for the diagnosis of

blood parasite infections
• Microscopic examination of blood
films is used
– malarial parasites,
– trypanosomes, and
– microfilariae.
The preparation of both

wet mounts (microfilariae and trypanosomes)
and
permanently stained
thick and
thin blood films is the mainstay of
diagnosis.
In the thin film
• The blood is spread over the slide

in a thin (single cell) layer, and the
red blood cells remain intact after
staining.
Thick films
• Thick films allow a larger amount
of blood to be examined, which
increases the possibility of
detecting light infections.
• Proper use of this technique
usually requires a great deal of
expertise and experience.
• In the thick film, the red cells are lysed before

staining, and only the white blood cells,
platelets, and parasites (if present) are
visible.
Blood films must be stained
• Giemsa or wright
stain is particularly
useful for the
staining of protozoa
Immunodiagnostics methods
• Detection of specific antibody responses to
the presence of the parasite.
– Demonstration of antibody can rarely differentiate

between acute and chronic infection.
• The measurement of circulating parasite

antigen in serum, urine, or feces may
provide a more appropriate marker for the
presence of active infection and may also
indicate parasite load.
Molecular Diagnostic Approaches
• Polymerase chain reaction provides sensitivity, allowing the
detection of as little as one organism in a biologic sample.
• This approach takes advantage to distinguish among strains,

species, and genera.
– Thus parasites may be simultaneously detected and identified in clinical
material
• Another advantage of nucleic-acid-based detection systems is that

they are independent of the patient's immunologic status or previous
infection history, thereby identifying active infection.
Animal Inoculation
• This approach is not practical for
most diagnostic laboratories……
• Animal inoculation is a sensitive

means of detecting infection caused
by blood and tissue parasites such
as
T. b. Gambiense
T. b. Rhodesiense
T. Cruzi
Leishmania species
T. gondii.
https://www.pexels.com/photo/beautiful-bird-bloom-blossom-414181/
STERILIZATION AND
DISINFECTION
Prof. Dr Tuba Dal

Yıldırım Beyazıt University
Faculty of Medicine
DEFINITIONS
🞆 Sterilization
A physical or chemical process that completely
destroys or removes all microbial life, including spores.
🞆 Disinfection
It is killing or removing of harmful microorganisms
🞆 Disinfectant
Products used to kill microorganisms on inanimate
objects or surfaces.
🞆 Disinfectants are not necessarily sporicidal, but may be

sporostatic; inhibiting germination or outgrowth
🞆 Antiseptic
A product that destroys or inhibits the growth
of microorganisms in or on living tissue.
🞆 Aseptic
Characterized by the absence of pathogenic
microbes.
RESISTANCE OF
MICROORGANISMS
Spores Sterilization
bacterial, fungal
Bacillus stearothermophilus
Bacillus subtilis
Clostridium sporogenes
Mycobacteria, TB bacilli
High Level Disinfection
Hydrophilic viruses Intermediate Disinfection
Polio, Coxsackie, Rhino
Vegetative fungi & bacteria

Low Disinfection
Lipophilic viruses
Trichophyton, Cryptococcus,Candida
Pseudomonas, Staphylococcus,Salmonella
HSV, CMV, RSV, HBV, HIV
🞆 Critical instruments are those used to
penetrate soft tissue or bone, or enter
into or contact the bloodstream or
other normally sterile tissue.
🞆 They should be sterilized after each
use.
🞆 Forceps, bone chisels, scalers and
surgical burs.
🞆 Semi-critical instruments are those
that do not penetrate soft tissues
or bone but contact mucous
membranes or non-intact skin
🞆 These devices also should be
sterilized after each use
🞆 3) Non-critical instruments are those that come into contact only
with intact skin such as external components of x-ray heads, blood
pressure cuffs .
METHODS OF STERILIZATION
1. Physical methods
⚫Heat
🞆 Dry
🞆 Moist
⚫Radiation
🞆 U.V. light
🞆 Ionizing radiation
⚫ Filtration
2. Chemical Methods
🞆 Sterilization by Heat: Most common
method
🞆 Dry Heat
⚫ Simplest method is exposing the item to the
naked flame e.g.
⚫ Bunsen burner- bacteriological loops, knives,
blades.
⚫ Hot air oven expose items to 160°C for 1
hour.
⚫ It has electric element in the chamber as
source of heat plus a fan to circulate air for
even distribution of heat in chamber.
⚫ Oven without fan is dangerous
⚫ Used for Metals, Glassware, Ointment, Oils,
Waxes, Powders i.e. items that are lacking
water
Sterilization Control of Hot Air Oven
• The spores of non-toxigenic strain of Bacillus subtilis and
Clostridium tetani are used as a microbiological test of dry heat.
• Browne’s test strip available that contain a chemical indicator.

Moist Heat: Uses hot water.

Moist heat kills microorganisms by denaturing proteins
Boiling – quite common especially in domestic

circumstances
Tyndallization
🞆 Lengthy process designed to reduce the level of activity
of sporulating bacteria
Moist heat:
Tyndallization
🞆 The process involves
🞆 boiling for a period (typically 20-30 minutes) at atmospheric
pressure,
🞆 cooling,
🞆 incubating for a day,
🞆 boiling,
🞆 cooling,
🞆 incubating for a day, boiling, cooling, incubating for a day,
and finally boiling again.
🞆 This technique is helpful in sanitizing heat-

sensitive culture media containing such materials
as sugars, egg or serum. which are harmed by
higher temperature of autoclave
🞆 Principle
🞆 Vegetative cells and a few spores are killed during the first warming and that the more safe spores
subsequently develop and are killed amid either the second or the third warming. In spite of the fact that for
the most part sufficient, this technique may fall flat with spores of specific anaerobes furthermore,
thermophiles.
Moist heat:
Pasteurization
🞆 It aims to reduce the number of viable pathogens in
liquids so they are unlikely to cause disease
🞆 It uses heat at temperatures sufficient to inactivate
harmful organism in milk.
🞆 Temperature may be 138°C for a fraction of a second

(flash method), 71.7°C for 15-20 seconds or 62°C for
30 minutes
Moist heat:
Autoclaving – Standard sterilization method in
hospitals.
🞆 The Autoclave works under the same principle

as the pressure cooker where water boils at
increased atmospheric pressure
🞆 The autoclave is a tough double walled chamber
in which air is replaced by pure saturated
steam under pressure.
Autoclaves, or steam sterilizers essentially consist of following:
i) A cylindrical or rectangular chamber, with capacities ranging from 400 to 800
liters.
ii) Water heating system or steam generating system
iii) Steam outlet and inlet valves
iv) Single or double doors with locking mechanism.
v) Thermometer or temperature gauge
vi) Pressure gauges
🞆 The usual temperature achieved is
121 °C at a pressure of 15 pps.i. at
exposure time of only 15-20 mins. By
increasing the temperature, the
time for sterilizing is further
reduced.
The pound per square inch or, more

accurately, pound-force per square inch is
a unit of pressure
Advantages of Autoclave
🞆 Temperature is > 100°C therefore spores are killed.
🞆 Condensation of steam generates extra heat (latent

heat of condensation).
🞆 The condensation also allows the steam to penetrate

rapidly into porous materials.
🞆 Note: that autoclavable items must be steam

permeable !!
Monitoring of autoclaves
🞆 Physical- use of thermocouple to
measure accurately the temperature.
🞆 Chemical- it consists of heat

sensitive chemical that changes color
at the right temperature and
exposure time.
⚫ Autoclave tape
⚫ Browne’s tube.
🞆 Biological – Spores of Geobacillus

stearothermophilus are used as the
test organisms as it is toughest
organism for an autoclave to destroy.
🞆 Its spores require an exposure of 15
mins at 121 C to be destroyed.
Sterilization by Chemical Methods

🞆 Useful for heat sensitive materials e.g. plastics and
lensed instruments endoscopes
🞆 Ethylene Oxide Chamber:
⚫ Ethylene oxide alkylates DNA molecules and thereby
inactivates microorganisms.
⚫ Ethylene oxide may cause explosion if used pure so it is mixed
with an inert gas e.g. Neon, Freon at a ratio of 10:90
⚫ It requires high humidity and is used at relative humidity 50-
60% Temperature : 55-60°C and exposure period 4-6 hours.
🞆 Activated alkaline Glutaraldehyde 2%:
⚫ Immerse item in solution for about 20 minutes if organism is
M.tuberculosis.
⚫ In case of spores, the immersion period is extended to 2-3
hours.
IRRADIATION
Radiation used for sterilization is of two types
1. Non-ionizing radiation, e.g. ultraviolet light, and
infrared light.
These forms of radiation can be used to kill or
inactivate microorganisms.
2. Ionizing radiation, e.g., X-rays, gamma rays, and
high speed electrons .
Non-ionizing radiation
⚫A. U.V. light- Has limited sterilizing power

because of poor penetration into most materials.
Generally used in irradiation of air in certain
areas eg. Operating Rooms and Tuberculosis
laboratories.
⚫B. Infrared –
⚫ It is most commonly used to purify air, such as
in the operating room. Infrared is effective,
however, it has no penetrating ability.
⚫Ionizing radiation
⚫ e.g. Gamma radiation: Source Cobalt60 has

greater energy than U.V. light, therefore
more effective.
⚫ Used mainly in industrial facilities e.g.
sterilization of disposable plastic syringes,
gloves, specimens containers and Petri Dishes.
METHODS OF
STERILIZATION
🞆Filtration
⚫May be done under either negative or
positive pressure.
⚫Best known example is the membrane
filter made from cellulose acetate.
⚫Generally removes most bacteria but
viruses and some small bacteria e.g.
Chlamydias & Mycoplasmas may pass
through.
⚫Thus filtration does not technically
sterilize items but it is adequate for
circumstances under which it is used.
⚫Main use: for heat labile substances e.g.
sera, antibiotics.
The recommended size filter that will exclude the

smallest bacterial cells is 0.22 micron
DISINFECTANTS
🞆 Factors influencing activity of Disinfectants

⚫ Directly proportional to temperature.
⚫ Directly proportional to concentration up to a point –
optimum concentration.
⚫ Time: Disinfectants need time to work.
⚫ Range of Action : Disinfectants are not equally
effective against the whole spectrum of microbes.
⚫ e.g. Chlorhexidine is less active against GNB than
Gram Positive Cocci.
⚫ May be inactivated by
🞆 Dirt, organic matter
🞆 Proteins, Pus, Blood, Mucus, Faeces
🞆 Hypochlorites and Glutaraldehyde are more
active against hepatitis viruses than most other
disinfectants.
DISINFECTANTS
Types of Disinfectants
Phenol and phenolics
🞆 Phenol (carbolic acid) is seldom used today.
🞆 Derivatives of the phenol molecule, however, are widely used.
🞆 Phenolics injure plasma membrane, inactivate enzymes, or
denature proteins. They are stable, persistent, and are not
sensitive to organic matter.
O-Phenylphenol
🞆 It is the main ingredient in most formulations of Lysol.
Hexachlorophene
🞆 It is used in nurseries and for surgical and hospital microbial
control procedures to control gram positive skin bacteria such
as staphylococci and streptococci.
🞆 Excessive use can cause neurological damage.
Triclosan
🞆 It is a widely used found in many household products.
🞆 It has broad spectrum of activity, especially against gram
positive bacteria, gram negative bacteria and fungi.
DISINFECTANTS
Biguanides
🞆 Chlorhexidine, a member of the biguanide group, is not a phenol, but its
structure and applications resemble hexachlorophene. It is frequently
used for surgical skin preparation and surgical hand scrubs.
Halogens
🞆 Iodine is effective against all kinds of bacteria, many endospores, fungi,
and some viruses.
🞆 Its mechanism of activity may be its combination with the amino acid
tyrosine in enzyme and cellular proteins.
🞆 An iodophore is a combination of iodine and an organic molecule.
🞆 Iodophores do not stain and are less irritating than iodine. Examples are
Isodine and Betadine.
🞆 Chlorine is used as a gas or in combination with other chemicals. Chlorine
gas is used for disinfecting municipal water supplies, swimming pools, and
sewage. Sodium hypochlorite – ordinary household bleach- is good
disinfectant.
🞆 Chloramines consist of chlorine and ammonia. They are more stable than
most chlorine. ……….tablets for field disinfection of water….
🞆 Chlorine dioxide in gaseous form is used for area disinfection, most
notably to kill endospores of anthrax bacteria.
DISINFECTANTS
Alcohols
🞆 Both ethanol and isopropanol (rubbing alcohol) are
widely used, normally at a concentration of about
70%. **
🞆 Concentrations of 60% to 95% are effective.
🞆 They are bactericidal and fungicidal but are not

effective against endospores or non-enveloped
viruses. ******
🞆 Alcohols enhance the effectiveness of other

chemical agents.
DISINFECTANTS
Heavy metals and their compounds
🞆 Tiny amount of heavy metals (e.g. silver and copper) are effective
antimicrobials.
🞆 1% silver nitrate solution has been used to prevent gonorrheal
ophthalmia neonatorum
🞆 Silver-sulfadiazine is used in wound dressings. Available as

topical cream for use on burns.
🞆 Mercuric chloride is highly bactericidal, but is toxic and

corrosive and is inactivated by organic matter.
🞆 Organic mercury compounds such as Mercurochrome are less
irritating and less toxic than inorganic mercury.
🞆 Zinc chloride is used in mouthwashes, and zinc oxide is used in

paints as antifungal.
Enjoy
the
Spring
…

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References

Murray et al. Medical Microbiology, 2020

Humans have an intimate relationship with microorganims; more than

Microorganisms are used in the production of fermented

Not all microorganims

Five kingdoms based on

1. Kingdom Prokaryotae (Monera):

TWO OF THESE THREE DOMAINS INCLUDE PROKARYOTEIC CELLS

1. Eubacteria: “True bacteria”.

Modern taxonomic approaches have recognized that some

Slime molds were formerly classified as fungi but are no longer

Although not forming a single monophyletic clade, they are grouped

Binomial nomenclature: Each organism (species) has

Klebsiella Honors Edwin Klebs The disease

Salmonella Honors Daniel typh- in mice (muri-)

Morphological characteristics Useful for identifying eukaryotes

Differential staining Gram staining, acid-fast staining

Biochemical tests Determines presence of bacterial enzymes

Biotyping: The use of biochemical tests to identify clinically

– Isolate is a general term for a pure culture of bacteria obtained

Culture: Grown in laboratory media

Bacterial Strain (A subgroup of a bacterial species ):

Clone: Population of cells derived from a single cell

➢ Define the cell wall in prokaryotes

➢ Describe the functions of cell wall

➢ Determine the differences of cell wall between gram

➢ Describe the functions of cytoplasmic membrane

➢ Define the bacterial cytoplasm and internal structures :

➢ The microscopic examination of clinical specimens is used to detect

➢ Characteristic morphologic properties can be used for the preliminary

➢ The microscopic detection of organisms stained with antibodies labeled

• Total power of magnification= the power of the objective X the

➢ Also known as resolving power

⚫ Under ideal conditions, resolving power of the light microscope is

⚫ With yellow light of a wavelength of 0.4 nm, the smallest

⚫ Shorter wavelengths provide a better resolution

⚫ Oil immersion lenses increase the numerical aperture

➢ The specimen produces an image that is brightly illuminated

➢ It does not allow for visualization of fine internal details of cells

➢ This technique has been particularly useful for observing organisms

➢ Allows differentiation of internal

➢ Useful for viewing intracellular structures

⚫ The image is colorful and

⚫ Allows for detailed view of live,

➢ Most often used on fluorescently

➢ Allows scientists to view the finest structure of

➢ Two forms: transmission electron microscope

about 2 micrometres (μm) long and 0.5 μm in diameter

⚫ The average diameter of the Erythrocytes is ~7 µm

charged) with a colorless anion

⚫ Eg. Gram’s staining, Acid-fast staining.

➢ 3. Special staining – more than one dye used -

➢ Gram(-) bacteria have a thin peptidoglycan

D)Peritrichous bacteria have flagella projecting in all directions (e.g., E. coli)

tuft at one end,lophotrishous all around bacteria,peritrichous

➢ Capsule is well organized layer and not easily washed

➢ Sticky web of polysaccharides in glycocalyx binds the cells together and to

➢ Biofilm is composed of numerous numbers of microorganisms

➢ Biofilm forms at interfaces of Plastic surfaces or prosthetic devices

➢ Most prokaryotes have peptidoglycan