General Microbiology & Genetics

Medical microbiology:
The science which is concerned with the study of microbes and the interaction
between microbes and hosts leading to infectious diseases.

Microbes are living organisms that can only be seen by microscopes and they are
sometimes called microorganisms although viruses are not true organisms.

Microorganisms

Prokaryotes Eukaryotes
Pro = premature Eu = true

Differences between prokaryotes and eukaryotes

Prokaryotes
Eukaryotes
(e.g. bacteria)
1-Nuclear membrane No nuclear membrane Present
2-Chromosome Haploid Diploid
3- Ribosomes 70 S (bacteria) 80 S
4- Division No mitosis Mitosis
5- Cell wall Peptidoglycan Absent
6- Examples Bacteria, viruses Fungi

Classes of pathogenic microorganisms

Viruses Bacteria Fungi
Size 0.02-0.2 μM 1-5 μM 3-10 μM
Nucleus ---- Nuclear body True nucleus
Ribosome ---- 70 S 80 S
Nucleic acid RNA OR DNA DNA + RNA DNA+RNA
Replication Intracellular Binary fission. Budding or mitosis.
Motility ---- Some ----
Cell wall Protein or lipoprotein Peptidoglycan Chitin
Intracellular and Intracellular and
Growth Intracellular
extracellular extracellular

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Bacterial Morphology

 Size of bacterial cell:

 Measured by micron (µ=micron= 1/ 1,000 mm).
 Gram staining:
 Bacteria are stained with Gram stain that can differentiate bacteria into: Gram
positive bacteria appearing violet or Gram negative bacteria appearing pink.
 Shape of the bacterial cell:
1. Cocci or spherical: e.g.
Staphylococci.
2. Bacilli or cylindrical: e.g.
Diphtheria bacilli.
3. Spiral:
 One curve: e.g. Vibrio.
 More than one: e.g.
Spirochetes, Spirillum.
 Arrangement:

Single Cluster Tetrads Pairs Chain angular

Bacterial structure

The basic components of bacterial cells include:

1- Cell envelope:
 Cell wall.
 Cytoplasmic membrane.
 Capsule.

2- Cytoplasmic components:
 Nuclear body.
 Plasmid.
 Ribosomes.

3- Cell appendages:
 Flagellae.
 Fimbriae "Pilli“.

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Cell Envelope
(1) Cell wall:
Chemical structure:
 The principal structural component of cell wall is peptidogycan.
Differences between Gram positive & Gram negative bacteria cell wall:
Gram positive Gram negative
Peptidoglycan Thick Very thin
Special structures  Teichoic acid  Lipoprotein
 Polysaccharides  Outer membrane
 Periplasmic space
 Lipopolysaccharise (LPS): "endotoxin"

Functions of cell wall:

1- Provides protection.
2- Maintain the shape of the
bacterial cell.
3- Contain endotoxin in Gram
negative bacteria.
4- Its polysaccharides and proteins
are antigens.

(2) Cytoplasmic membrane:

 It is a thin elastic membrane lies
immediately under the cell wall.
Nature:
 Protein and phospholipids.
Functions:
1) Nutrients transport.
2) Electron transport for energy
production.
3) Synthesis of cell wall components.

(3) Capsule:
 It is gelatinous material surrounding bacterial cells produced by some pathogenic
bacteria inside the host tissue.
Chemical structure:
 Complex polysaccharides e.g. Streptococcus pneumoniae.
 Polypeptides e.g. bacillus anthracis.
Functions:
1. Antiphagocytosis.
2. Virulence.
3. Antigenicity: “K” antigen.

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Cytoplasmic components
(1) Ribosomes:
 The factory of protein synthesis in the cell.
 Complex structures composed of RNA & proteins.
 It is a target for some antibiotic as tetracycline and chloramphenicol.
Bacterial ribosomes consist of (70S):
Large subunit: 50S.
Small subunit: 30S.
S value = Svedberg unit.

(2) Nuclear body (Nucleoid):

 No nuclear membrane.
 DNA molecules folded on itself.
 Single chromosome is double stranded circular DNA carries genetic characters.

(3) Plasmids:
 Extrachromosomal genetic elements.
 Circular double stranded DNA.
 Carry certain function: antibiotic resistance.

Cell appendages
1- Flagellae:
 They are long helical filaments attached to cytoplasm and pass out the cell wall.
 Formed of contractile protein "Flagellin".
Demonstration:
1) Motility test: Hanging drop.
2) E/M.
Functions:
1) Motility:
2) Antigenicity: H antigen.

2- Fimbriae (Pili):
 They are short, hair like filaments.
 They are shorter and thinner than flagella straight and not originating from cytoplasm.
 They are formed of protein "pilin".
Functions:
1) Organs of adhesion to host cell "common pili".
2) Sex pili “conjugation”.
3) Virulence.
Bacterial spores
 Bacterial endospores are resting body phase formed under unfavorable conditions. They are
highly resistant forms produced outside the body by some organisms like Bacillus and
Clostridium groups.

Shape: Spores may be spherical or oval.

Site: They may be Central, sub-terminal or terminal.

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Bacterial Products
(1) Bacterial enzymes:
Types:
 Proteolytic enzymes: act on proteins.
 Saccharolytic enzymes: act on carbohydrates.
 Lipolytic enzymes: act on lipids.
 Respiratory enzymes.

(2) Bacterial pigments:

a- Endopigment:
 Localized inside the organism and giving color to the colony.
 Example: Golden yellow pigment of Staphylococcus aureus.

b- Exopigment:
 Diffuses from the organism giving color to surrounding medium.
 Example: the bluish green pigment of Pseudomonas aeruginosa.

(3) Bacterial toxins:

1. Exotoxin 2. Endotoxin
Diffusibility Diffusible Cell bounded
Antigenicity Strong Weak
Toxicity High Low
Specificity Specific action on cells Non specific
Nature Protein Lipopolysaccharide
Source Gram positive & some negative Cell wall of Gram negative
Heating Labile Stable
Effect of formalin Change to toxoid Not affected

Growth Requirements of Bacteria

(1) Nutrition:
 Basic elements: Carbon and Nitrogen.
 Major elements: Phosphorus and Sulpher.
 Minor elements: Mg ++, K+, Ca ++ and Iron.
 Essential metabolites and growth factors like Nucleotides and vitamins.

(2) Gases:
►Oxygen:
(1) Obligatory aerobes: Grow only in the presence of O2 e.g. Mycobacterium
tuberculosis.
(2) Facultative anaerobes: Grow well in the presence and absence of O2 e.g. pathogenic
bacteria.

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(3) Obligatory anaerobes: they can not grow in the presence of O2 e.g. Clostridium.
(4) Microaerophilic: They grow best in the presence of a minimal amount of O2.

►CO2:
 The normal atmospheric CO2 (0.03%) is usually sufficient for growth of most bacteria.
 Some organisms may require higher CO2 concentration e.g.: for stimulation of growth or
toxin production.

(3) Temperature:
 The optimum temperature for growth and multiplication of most pathogenic bacteria is
37oC.
 They have a minimum temperature (10oC) below which they can not grow and a maximum
temperature (42oC) above which they can not grow.

(4) Moisture.
(5) Hydrogen ion concentration (pH):
 Pathogenic bacteria grow at a narrow range of pH with an optimum 7.5.
 Some bacteria need alkaline pH e.g. Vibrio cholera.
 Some bacteria need acidic pH e.g. Lactobacillus acidophilus.

Bacterial Growth and Reproduction

Growth:
 Increase in the number of bacterial cells which result from increased in biomass of bacteria.
Reproduction: Simple binary fission
Steps:
1- Increase in size.
2- Elongation.
3- Transverse septum.
4- Dublication of chromosome.
5- Formation of new cells.

Bacterial metabolism
Metabolism consists of two concomitant processes.

Catabolism Anabolism
 Break down of CHO & lipid & proteins  Synthesis of cellular component from
(oxidation) into precursors precursors

 Variable in bacteria  Similar in bacteria

 Produce energy (lost as heat& stored)  Needs energy

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Bacterial Growth Curve

 When bacteria are cultured on fluid media, the viable bacterial number follows a course
called "growth curve".
 The growth passes into four stages when we plot the logarithmic number of viable bacteria
against time in hours.

Steps:
a) The lag phase.
b) The logarithmic phase.
c) The stationary phase.
d) The decline phase.

1. Lag Phase:
 No increase in bacterial numbers as bacteria prepares themselves for active division.
 The duration of the lag phase may be few hrs to few days depending on:
1- Type of organism: E. coli < 1hr, while T.B. bacilli: few days.
2- The stage of the inoculum.
3- Medium (suitable).
 It correspond to the incubation period of the disease.

2. Logarithmic Phase:
 Division occurs at a maximum rate (active & regular division).
 Represented as ascending straight line.
 Bacteria keep dividing rapidly till a point (saturation point) which depends on type of
organism and environmental factors (suitability of the medium and the growth conditions).
 This stage corresponds to the invasive period of disease.

3. Stationary Phase:
 In this stage the rate of division = rate of death so the number of living organisms remains
stationary.
 Growth rate decrease due to:
1- Exhaustion of food.
2- O2 starvation.
3- Accumulation of toxic materials.
 This stage corresponds to the period of signs and symptoms of disease.

4. Decline phase:
 In this stage the rate of death > rate of growth and at end, bacteria are completely died.
 Increased death rate is due to:
1. Accumulation of toxic metabolites.
2. O2 exhaustion.
 This phase corresponds to the convalescent period of the disease.

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Bacterial virulence factors

The following table summerize the most important studed virulence factors:
Virulence factor Function
1- Capsule: - Antiphagocytic
- Adherence
Colonization

- Protect against complement and lysozymes

2- Adherence factors:
- Cell surface proteins - Bind to specific epithelium receptors

- Pili (fimbriea) - Promote colonization

3- Enzymes: Collagenase: degrade collagen
s Toxigenesi Invasion

For invasion and spread Hyaluronidase: degrade hyauronic acid

Coagulase: clot plasma and protect from
Endotoxin -phagocytosis
Similar action:
andfever, hypotension,
antibodies
hypoglycemia, hemorrhage and shock
Exotoxin - Different toxin classes (neurotoxins,
Ability to survive intracellular Escape host immune defense

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Microbial Genetics

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Microbial Genetics


Genetics is the science concerned with the cell characteristics, and how they are
passed from one generation to the next.
Gene: it is the unit of heredity. It is a segment of DNA that carries, in its nucleotide
sequence, information for specific biochemical or physiologic property.
Phenotype: All the heritable physical characters of the cell that are controlled by the
genotype (eye color in human, resistance to antibiotic in bacteria ….. etc.).
Genotype: It means the information in the DNA that controls the phenotypes.

:Molecules of Genetics

 The main molecules of genetics are called nucleic acids and all the genetic information is
stored as a sequence of bases through these molecules mainly in DNA and some RNA
viruses.
1- DNA (Deoxyribonucleic acid ):
 It serves as an organism’s genetic material.
 It is divided into functional units (genes).
 Most of DNA is double stranded.
 It consists of non-identical, complementary base sequences.
 The two strands are held together by hydrogen bonds between adenine and thymine or
guanine and cytosine.
 The basic structure of DNA molecules are of three components:
o Sugar:
 It is a cyclic form of 2-deoxyribose sugar that forms the backbone of the DNA.
o Nitrogenous bases:
 It is the cyclic structure of purine and pyrimidine rings.
o Phosphates:
 Sugar back bone are linked to each other by phosphodiester bonds, i.e., a single
phosphate connected by ester linkage to two sugars; it means that DNA
molecule consists of altering units of phosphate and 2- deoxyribose.
 The phosphate connects two sugars by bonding with the 3' carbon of one sugar
and with the 5' carbon of the next sugar.

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DNA
Structure

 DNA is only polymerized 5' to 3' and as antiparallel i.e. one strand in the direction 5'→3'
and the other strand polymerized in the opposite direction.
 Most of DNA molecules in prokaryotes are double helix and in circular manner.
 This circular double stranded DNA molecules are twisted and compacted through a super
coiling process this is done naturally by topoisomerase enzymes.

2- RNA (Ribonucleic acid):

 Structurally similar to DNA except:
o Most of RNAs are single stranded.
o Sugar is ribose instead of deoxyribose.
o Uracil base instead of thymine base.
 Functionally different than DNA:
o Some of RNAs are used as messenger
molecules to transfer information from
DAN to protein (mRNA).

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o Some of RNA as a part of ribosomes (rRNA).

o Some are adaptor molecules (tRNA).
o Few RNA only acts as a genetic material like DNA (the viruses).

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DNA replication in bacteria

 In 1953, James Watson and Francis Crick presented a molecular model of the
double- helix structure for DNA.
 This depends upon this model and the complementarity between G≡C and A=T.
 The most accepted theory that replication is a semi conservative manner it means
that one DNA molecule gives two DNA molecules each one consists of one strand
from the original DNA and the other strand is newly formed one.
Direction of replication:
 Replication starts at a fixed point called origin of replication (ori) in E. coli called
(oriC) and terminated at fixed point called (ter) point.
 Under optimum conditions each replication fork moves at a rate of 970 bp/sec.
Steps of replication:
1. The two old strands of DNA are separated by helicase enzyme to form the
replication fork.
2. Replication of one strand template in 5' to 3' direction can proceed in a continuous
fashion from 5' to 3'; this is called the leading strand.
3. Replication of the other template strand can only proceeds discontinuously because
its direction is from 3' to 5' as fragments called Okazaki fragments.
4. After formation of the two strands of DNA, the DNA gyrase enzyme
(Topoisomerase II) makes twist and super helicity of each DNA molecules.

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Gene Expression
The genetic information in DNA is expressed by copying DNA into RNA and the RNA is
translated into a protein. Genetic information flows from DNA to mRNA to proteins.

a) Transcription:
 It is the process by which a single stranded RNA is formed by RNA- polymerase using
DNA (gene) as a template this RNA is called messenger RNA (mRNA).
 The mRNA has a nucleotide sequence complementary to a template strand in the DNA
double helix if read from 3' to 5' directions
 The sequence of events occur as following:
1) Promoter recognition (area at DNA recognized by RNA polymerase before the gene
to be transcribed on which RNA – polymerase become attached).
2) Chain initiation in which one recognition protein called sigma factor (σ) attached to
RNA polymerase to put it at the correct site of the first nucleotide to be Chain
elongation in which RNA
polymerase synthesizes the whole
length of mRNA by polymerizing
the complementary bases in the
5'-3' direction (note: RNA
polymerase does not need primer
to start unlike DNA polymerase).
3) Chain termination, RNA
polymerase continues until a
transcription termination signal
or terminator where the
polymerase and newly Transcriptio
synthesized RNA dissociate from n
the DNA to end the transcription.

b) Translation (Reading the code):

 Protein synthesis occurs on ribosomes.
 The process is started by attachment of
small ribosome subunit carrying a
methionine tRNA with the mRNA at the
starting codon (AUG).
 Once the initiation complex has formed,
synthesis of polypeptide chain is driven by
elongation factor that join the large subunit
(50S) of the complex and move the
ribosome relative to the mRNA.
 Each tRNA carries an amino acid and triplet of bases (anticodon) which recognize a
codon on mRNA, for example: tRNA that carries methionine has anticodon UAC that
recognize the codon AUG.

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Polymerase Chain Reaction (PCR)

 PCR is a technique for making millions of copies of a specific target sequence of
nucleotides in DNA.
Requirments:
(i) The sample dsDNA
(ii) The primers: small pieces of ssDNA each about 20-30 nucleotides in length.
(iii) Deoxyribonucleoside triphosphates of all four types
(iv) DNA polymerase.

Steps:
The above mentioned reaction mixture undergoes a series of changes in temperature in a
thermocycler device. This cycle is repeated about 20-35 times. Each cycle includes:

1- Denaturation step: initial heating to about 94ºC denatures the dsDNA fragment to two
single strands.
2- Annealing step: transient cooling to 45- 60ºC allow the primers to bind (anneal) to their
complementary sites on the sample DNA.
3- Extension step: a change to 72ºC then permits the DNA polymerase enzyme to start
DNA amplification from the 3‘ end of each primer. When the temperature cycle is
repeated, the newly synthesized strands act as templates and so on.

Uses of PCR:

1. For diagnosis of:

o Microbial disease: bacteria, viruses or fungi.
o Genetic disease.
2. Medico legal purposes.
3. Typing of microorganisms.
4. Taxonomy and evolutionary purposes.

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Anti-Microbial Agents

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Anti-Microbial Agents
 Definition: Anti-microbial agents include antibiotics, anti-viral and anti-fungal drugs.
 Mechanism of action of clinically used antibiotics:
1-Inhibition of cell wall synthesis.
2-Alteration of cell- membrane permeability.
3-Inhibition of protein synthesis.
4-Inhibition of nucleic acid synthesis.
5-Other mechanisms of action.
1. Antimicrobial action through inhibition of cell wall synthesis:

Cell wall inhibitors

B- Lactams Polypeptides
Glycopeptides

Penicillins Cephalosporins Monobactams Carbapenems

•Aztreonam •Imipenem

Mechanisms of action:
Penicillins and cephalosporins act through the inhibition of the terminal cross- linking of the
peptidoglycan.

 Resistance to penicillin:
1. The organism produce penicillin destroying enzyme ß- lactamases
2. Absence of penicillin receptors.

2. Antimicrobial action through inhibition of cell membrane function:

 Examples: Polymyxin, Nystatin.

 Mechanisms of action:
Distortion of the protein and phospholipids layers of cell membrane and allow free passage of
substances inside and outside the cell.

3. Antibacterial action through inhibition of protein synthesis:

Examples: Aminoglycosides (Amikacin, Gentamicin).

Tetracycline.

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Mechanisms of action:
a) Aminoglycosides: They inhibit translation in the 30S of ribosome.
b) Tetracycline: they block the action of 50S
4. Antimicrobial action through inhibition of nucleic acid synthesis:

Examples and mechanism of action:

a) Sulphonamides act by competitive inhibition with para-amino benzoic acid (Precursor of
nicotinic acid). The later is precursor of the synthesis of purines → DNA.
b) Trimethoprim: also inhibit the oxidation of nicotinic acid in the synthesis of purines.
c) Rifampicin: Inhibits mRNA synthesis.

Causes of failure of antimicrobial chemotherapy:

(1) Clinical condition not suitable to antibiotic treatment (as viral infection or mixed
bacterial infection).
(2) Failure to use laboratory properly.
(3) Wrong choice of antibiotics.
(4) Inadequate doses of the antibiotics.
(5) Inadequate duration of treatment by the antibiotics.
(6) Wrong route of administration.
(7) Use of antagonistic antibiotic combination.
(8) Development of antimicrobial resistance.

Drug resistance
- It is the unresponsiveness of the organisms to the given drug (antibiotics).
Mechanisms of Drug resistance:
1) Microorganisms produce enzymes that destroy the drug: as β- lactamases enzymes produced
by Staphylococci to destroy the β-lactams.
2) Microorganisms decrease their permeability to the drug.
3) Microorganisms develop an altered structural target for the drug: As alteration of the
receptor protein that give attachment to the drug.
4) Microorganisms develop an altered metabolic pathway that bypasses the reactions inhibited
by the drug.
5) Microorganisms develop an altered enzyme that can still perform its metabolic function but
is much less affected by the drug.

Origin of drug resistance:

- The origin of drug resistance may be:
a) Non- genetic: in this case microorganism may loss the specific target structure for the drug
for several generations.
b) Genetic origin:
1- Chromosomal resistance: this results after spontaneous mutation in a locus that controls
susceptibility to antimicrobial drug (change receptors, permeability).
2- Extra chromosomal resistance (plasmids):
 Plasmids genes for antimicrobial resistance often control the formation of enzymes
capable of destroying the drug.

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