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Medical Microbiology
Prof. Dr. Jie YAN (严杰)
Department of Medical
Microbiology and Parasitology
E-mail: [email protected]
School of Medicine
Zhejiang University
Introduction to medical microbiology
Microbes / Microorganisms
•The word “microbe” comes from the Greek words mikros,
meaning small life. So microbes / microorganisms are small
living things that are too small to be seen by naked eye.
•Microorganisms were probably the first organisms to appear
on the earth.
•However, these organisms were not seen until about 3
centuries ago when lenses powerful enough to make them
visible were made.
•Viruses, bacteria, fungi, protozoa and some algae are all in
this category.
Distribution
•The distribution of microorganisms is universal in nature
including air, soil, water, animals and human body.
Relationship with human beings
•There is a close relationship between microorganisms and
human beings.
• Beneficial activities: Most microbes are benefit to human
beings, some are necessary (nitrogen and arbon cycles).
• Harmful activities: Only a small portion of microbes cause
human diseases, which called pathogenic microbes.
Medical Microbiology
• Medical microbiology is a branch of Microbilogy to study
biological character, pathogenicity and immunoty, laboratory
diagnosis, and prevention and control of pathogenic microbes.
Microbes in nitrogen cycle
Prokaryotes / Eukaryotes
• The prokaryotic cell, in contrast to the eukaryotic cell, has no nuclear
membranes, mitochondria, endoplasmic reticulum, Golgi body,
phagosomes and lysosomes.
• Prokaryotes generally possess only a single circular chromosome,
which is bound to a specific site on the cell membrane - the mesosome.
• Prokaryotic ribosomes are 70S (30S and 50S subunits) in size, whereas
eukaryotic ribosomes are larger (80S, 40S and 60S subunits).
Classification of microbes
•According to organizational structure, microbes can be
divided into three types:
Prokaryotes (Eubacteria and Archaebacteria)
Eukaryotes (fungi, Protozoa, algae)
Acellular entities (viruses)
•Eubacteria include Bacteria, Chlamydiae, Mycoplasmas,
Richettiae, Spirochetes, and Actinomycetes. Some of them cause
human diseases.
Viruses
•Viruses are very small particles and have no basic cell
structure. A simplest virus consists of one core and one
protein coat (capsid). The core composed with a nucleic acid
molecule, either DNA or RNA.
•Viruses are obligate parasites totally dependent on their host
cells for replication.
Fungi
•Fungi is a kind of eukaryotic cells. So they have various
organelles, for examples, nuclear membranes, mitochondria,
endoplasmic reticulum, Golgi body, phagosomes and lysosomes.
New challenge in medical microbiology
•The numerous emerging and re-emerging infectious diseases
such as AIDS, SARS, avian influenza, tuberculosis, viral
hepatitis and so on.
Bacteriology
Morphology and Structure of Bacteria
Size of bacteria
• Unit for measurement of bacteria is micrometer (μm)
• On the average, bacteria are 2-8 μm in length and 0.2-2.0 μm
in diameter. Exceptions include some spiral shaped bacteria
that can reach 4- 500 μm.
 1000
Shape of bacteria
• Spherical (Cocci, sing. Coccus )
• Rods (Bacilli, sing. Bacillus)
• Spiral (Spiral bacteria)
vibrio
spirillum
helicobacterium
Spherical bacteria
Different arrangements depending
on the plane of division
Diplococci: Pair of cells divide in one plane
Streptococci: Chain of cells formed by
dividing in one plane several times
Tetrad: Divide in two planes
Sarcinae: Divide in three planes
Staphylococci: Divide in many planes and
remain together as a cluster
Rod-shaped bacteria
•
Considerable variation in length
and diameter: 0.5-1 μm in width
and 2-5 μm in length.
•
Most of rod-shaped bacteria are
single arrangement.
Diplobacilli: Bacilli that remain
in pairs after they divide.
Streptobacilli: Bacilli that remain
in chains after they divide.
Coccobacilli: A short Bacilli that
nearly looks like a cocci.
Spiral-shaped bacteria
Divided into:
Vibrio: comma shaped
Spirillum: helical
Structure of Bacteria
• bacterial
structures may be defined:
Cell envelope
Plasmids
Flagella
Pili
Capsules
Spores
Important bacterial structures
Cell envelope
Plasmids
Flagella
Pili
Capsules
Sspores
Cell envelope
•Bacterial envelope is divided into cell membrane and cell wall
(Gram positive) plus an outer membrane (Gram-negative).
Gram-positive cocci
Gram-negative bacilli
(Gram-staining method)
Cell wall: general component-peptidoglycan
•Cell wall consists of peptidoglycan layer and attached structures.
Gram-positive
Gram-negative
Peptidoglycan
•glycan backbone: N-acetyl
muramic acid and N-acetyl
glucosamine are alternatively
linked by -1,4 linkage.
•4-peptide side chain: links
to N-acetyl muramic acid.
•peptide bridge: links side chains (gram-negative bacteria have
no peptide bridges).
•Penicillin can block the linkage between peptide side chain and
bridges to kill gram-positive bacteria.
Cell wall: characteristits of gram-positive bacteria
•Peptidoglycan layer is thick (15-50 layers).
•There are some special components such as teichoic acids, the
major superficial antigen of gram-positive bacteria .
Cell wall: characteristits of gram-negative bacteria
•Peptidoglycan layer is thin (1-2 layers).
•There is outer membrane located in outside of peptidoglycan
layer but no any teichoic acids.
Outer membrane
•Outer membrane of a gram-negative bacterium is composed of
phospholipids, membrane proteins and lipopolysaccharide (LPS)
Lipopolysaccharide (LPS)
•LPS is also called endotoxin (poisonous to mammal cells).
•LPS has 3 regions: an external O antigen, a middle core, and
an inner lipid A.
• O antigen is a polysaccharide
to act as the somatic antigen of
gram-negative bacteria.
•Core polysaccharide links O
antigen with lipid A.
•lipid A decides toxicity.
Cell wall: function
• Maintaining bacterial shape.
• Resistance to osmotic pressure
• Providing a platform for surface appendages such as
flagella and pili.
• Providing a pathogenic function to adhere host cells
(For gram-positive bacteria, the major adhesin is teichoic
acids. For Gram-negative bacteria, the major adhesin is pili
and some of outer mambrane proteins).
• Playing an essential role in bacterial division
• Participating bacterial material exchange
• Containing major antigens.
Wall-less forms of bacteria
• When bacteria are treated with 1) enzymes (e.g. lysozyme) with
cell wall hydrolytic activity or 2) antibiotics inhibiting
peptidoglycan synthesis, wall-less bacteria are generated which
is called L-forms of bacteria.
• L-forms of bacteria can cause chronic infections.
• L-forms of bacteria are difficult to cultivate and usually
require a medium with a right osmotic strength.
• It is resistant to antibiotics (e,g. penicillin) and difficulty to
detect (e.g. absence of O antigen).
Electron micrograph of Staphylococcus
A: L-form; B: wild type
Important bacterial structures
Cell envelope
Plasmids
Flagella
Pili
Capsules
Spores
Plasmids
•Plasmids are small, circular / line,
extra-chromosomal double-stranded
DNA.
•Usually present in multiple copies and
are capable of self-replication.
•Often code for pathogenic factors and
antibiotic resistant factors. Are not
essential for bacterial survival.
Important bacterial structures
Cell envelope
Plasmids
Flagella
Pili
Capsules
Spores)
Flagella: general description
• Flagellum is composed of flagellin and provide motility.
•It extends from cell envelope and projects as a long strand.
•Flagellum is slender that can not be seen by light microscopy
unless a special stain is applied.
Flagella: structure
• Basal body:
a structure to
insert into cell
envelope.
• Flagellin is
an antigen (H
antigens).
Flagella: function
•Motility of bacteria: move towards foodstuffs or away from
toxic materials.
•Identification of bacteria: According to the mobility and antigenicity
of flagellin (H antigen).
•Possible pathogencity: chemotaxis to the suitable sites in hosts
for colonization.
Important bacterial structures
Cell envelope
Plasmids
Flagella
Pili
Capsules
Spores
Pili
•Pili are hair-like strands of bacteria.
•They are shorter and thinner than flagella, only visible under
electron microscope.
Pili
•Pilus is composed of special protein called pilin.
•Two types can be distinguished:
Ordinary pili
•Shorter, thinner, numerous for a bacterium
•Relative to bacterial adhesion (adhering to host cells)
•Contribute to virulence of some pathogenic bacteria
Sex pili
•Longer, coarser, only 1-4 for a bacterium
•Relative to bacterial conjugation (a pattern of bacterial
genetic material exchanges)
•The recent data revealed the sex pili of some bacteria
has the ability to adhere host cells.
Ordinary pili
Sex pili
Recipient
Donor bacterium
Electron graph of pili
Important bacterial structures
Cell envelope
Plasmids
Flagella
Pili
Capsules
Spores
Capsules and slime layers
•Capsule is a structure surrounding outside of cell envelope.
•Usually, slime layer is thinner than capsule.
•They are usually demonstrated by the negative staining or
“capsule stain” which gives color to the background.
Capsules and slime layers
•They are usually composed of polysaccharide. However, in
some certain bacilli, they are composed of polypeptide.
• They are not essential to bacterial viability.
• Some strains within a bacetrial species can produce a capsule,
whereas the others can not.
•Capsules are often lost during in vitro culture.
•The capsules contribute to invasiveness (virulence) of bacteria
by protecting them from phagocytosis by phagocytes.
Important bacterial structures
Cell envelope
Plasmids
Flagella
Pili
Capsules
Spores
Spores
• Under adverse conditions, such as nutrient / water depletion,
some bacteria form a thick wall inside the cytoplasmic
membrane leading to a resting stage known as spores.
•Spores contribute to bacterial resistance.
Spores
•One spore-forming bacterium can only produce one spore
which has no propagation ability.
•One spore germinates into one vegetative bacterial cell which
can propagate / multiplication.
•Spore can be seen after staining with dyes. Sometimes, it can
also be seen as a colorless area by using conventional bacterial
staining methods.
•Spores are commonly found gram-positive bacilli.
•Different sizes, shapes and positions of spores will help us to
identify spore-forming bacteria.
Structure of spores
Core spore wall /core
Cortex Coat Exosporium
Classification of bacteria
•Taxonomic terms:
Family: a group of related genera.
Genus: a group of related species.
Species: a group of related strains.
Type: sets of strains within a species (e.g. biotypes, serotypes).
Strain: one line or a single isolate of a particular species.
•The basic taxonomic group is species.
strain  type  species   genus     family
O157:H7
Coli
Escherichia
Enterobacteriaceae
Classification of bacteria
Staphylococcus aureus
S. aureus
Genus
species
金黄色葡萄球菌
species genus
Summary
Structure of bacteria include essential structures of cell wall,
cell membrane, cytoplasm, and nuclear material (nucleoid).
Some bacteria also have one or more of the particular
structures of capsule, flagella, pili, endospores.
Structure of cell wall, cell wall structural differences
between Gram-positive and Gram-negative bacteria, concept
of plasmid, and functions of bacterial particular structures are
the most important contents, because of their close association
with bacterial pathogenesis.
Growth, Propagation and Metabolismof
Bacteria
Nutrtion types of bacteria
• Autotroph: can synthesize organic substances using CO2 as
carbon source and N2 or NH3 as nitrogen source. The
energy comes from oxidation of inorganic substances.
• Heterotroph: use different organic substances, such as
proteins, saccharides and lipids, as the nutrient substances
or materials and energy source.
▲Saprophyte: dead bodies of animals and plants, or
decomposed foods.
▲Parasite: living hosts (animals and/or human).
Nearly all the pathogens are parasites.
Nutrient substances of bacteria
• Water: mediator for biological responses.
• Carbon source
• Nitrogen source
• Inorganic salts: have many functions to act as a component
of organic substance as well as to maintain enzymatic
activity and osmotic pressure and pH, etc.
• Growth factors: vitamins, some special amino acids,
hemoglobin and coenzyme I or II (blood, serum) .
Conditions of bacterial growth and propagation
•
•
•
•
Nutrient substances
pH: 7.2-7.4 for microbial pathogens.
Temperature: 37ºC for microbial pathogens.
Gas: O2
▲Obligate aerobe: needs O2 during growth and propagation.
▲Microaerophilic bacteria: 5% O2.
▲Facultative anaerobe: grow and propagate in aerobic or
anaerobic enviroment.
▲Obligate anaerobe: has no special enzymes (e.g. SOD and
catalase) to deal with ROS such as O ¯2 and H2O2 produced
in metabolism.
Bacterial growth and propagation
• Growth and propagation of a bacterial individual: binary
fission (2n), a process in which a parent cell splits into two
daughter cells with approximately equal size.
a. Bacterial cell first can been seen to enlarge
or elongate.
a
b. Followed by formation of
membrane and new cell wall.
b
transverse
c. The new membrane and cell wall grow
inward from the outer layers.
d. The cell divided into the two daughter cells.
c
d
Bacterial growth and propagation
• Generation time: under optimal conditions, the average time
required for a population of bacteria to double in number.
20-30 min for most of bacteria (e.g. E. coli).
• Colony: a bacterial cluster from propagation of a bacterium.
▲Obtain a pure bacterial species.
▲Often used for bacterial counting.
Bacterial growth and propagation
• Growth and propagation of a bacterial population:
Growth curve
Stationary Phase
OD600
Log Phase
Death Phase
Lag Phase
Time
Bacterial growth and propagation
• Phenomena of bacterial growth in liquid medium
Broth (a common liquid medium)
cultures can exhibit: (i) forming
cloudiness in broth (growth with
uniform turbid pattern), or (ii)
forming a ring at the top of broth
(growth with suspension pattern),
or (iii) forming sediment at the
bottom of broth (growth with
sedimentary pattern).
i
ii
iii
Constructive metabolism of bacteria
• Pyrogen: cause fever (LPS of G- bacteria and glycopeptide
or glycolipid of G+ bacteria).
• Toxins: exotoxins and endotoxin (LPS).
• Invasive enzymes: e.g. collagenase (invasion and spreading)
and coagulase (resist phagocytosis of macrophages).
• Others: pigment, vitamine, antibiotic, bacteriocin (细菌素).
Destructive metabolism of bacteria
For identification of bacteria !
• Carbohydrate
Fermentation Tests
Positive: yellow color or
yellow color with gas bubble
Negative: red color and no
gas bubble
Destructive metabolism of bacteria
Methyl Red (MR) Test
hydrolyse pyruvate (丙酮酸)
Destructive metabolism of bacteria
Voges-Proskauer (VP) Test
hydrolyse pyruvate (丙酮酸) → diacetyl (二乙酰)
Destructive metabolism of bacteria
Citrate Utilization Test
The citrate test utilizes Simmon's
citrate media to determine if a
bacterium can grow utilizing citrate
as its sole carbon and energy
source.
Growth of bacteria in the media
leads to development of a Prussian
blue color (positive citrate).
Destructive metabolism of bacteria
Indole Test
hydrolyse tryptophan
to produce indole
Destructive metabolism of bacteria
Hydrogen Sulfide (H2S ) Formation Test
To determine the ability of a
bacterium to produce hydrogen
sulfide (H2S) by enzymatic reaction
on amino acids such as cysteine,
cystine
and methionine.
Positive result: The hydrogen
sulfide combines with ferrous
sulfide (Fe2S) in the triple sugar
iron (TSI) agar to form a black to
dark insoluble precipitate.
Destructive metabolism of bacteria
Urease Test
Principle: The hydrolysis of urea by urease
produces ammonia and carbon dioxide. The
formation of ammonia alkalinizes the
medium, and the pH is detected by the color
change from light orange to pink-red.
Positive result: pink-red color
Negative result: light orange
Death of Microorganisms
Disinfection & Sterilization
Concept and Definition
Sterilization: A physical or chemical process to kill all microbial
life including spores.
Disinfection: A physical or chemical process to kill vegetative
microbes, but not kill spores.
Bacteriostasis: A physical or chemical process to inhibit bacterial
growth / propagation in vitro and in vivo.
Antisepsis: A physical or chemical process to inhibit bacterial
growth / propagation in vitro, but not kill bacteria.
Asepsis: a state of being free of living microbes.
Antimicrobial agents
▲ Physical Agents: Heat, Radiation, Filtration,
Low Temperature and Desiccation (Dry)
▲ Disinfectants and Antiseptics
Physical antimicrobial agents: Heat
▲ A temperature of 100 ºC (boiling) usually for 2-5
min will kill all vegetative forms but not kill spores.
▲ A temperature of 121 ºC for 15-20 min will kill
all microorganisms including spores (autoclave).
▲ Hot air sterilization by hot air ovens, heating at
160 ºC for 2 h,
Physical antimicrobial agents: Ultraviolet Ray
▲ Microbial killing effect of sun light is due in large part to
the action of ultraviolet light.
▲ Activity of ultraviolet (UV) ray depends on: i) Length of
exposure: 30 min; ii) Wavelength of UV ray: 260 nm - 270 nm
thymine-thymine dimmers
within the one DNA strand
will block base pairing and
DNA replication.
Summary
The most important contents in this lecture are displayed as
the followings:
1) Bacteria growth curve, especially the characteristics and application of
log phase and maximum stationary phase.
2) Concepts of sterilization, disinfection and asepsis, and the temperature
and time to kill bacteria including spores when using autoclaving and
hot air sterilization.
3) The microbicidal mechanism and application limits of UV radiation.
4) The types (names) of bacteria based on the difference of O2
requirement in growth.
5) Concepts of bacterial colony, pyrogen and invasive enzymes.