My Microbiology Notes 2

Lect 7 

Microbial genetics

1. Genetic Material

DNA & RNA

DNA=deoxyribonucleic acid

RNA=ribonucleic acid 

Basic building blocks:

Nucleotides

Phosphate group

Pentose sugar

Nitrogenous base

2. Structure of DNA

 Double stranded (double helix) 

 Chains of nucleotides 

 5’ to 3’ (strands are anti-parallel) 

 Complimentary base pairing 

◦ A-T

◦ G-C

#Prokaryotic chromosome

3. DNA Replication

 Semiconservative

◦ Replication fork

 Single origin

 Bidirectional

 2 Leading strands

 2 Lagging strands

 Enzymes

 Helicases

 DNA polymerases 5’ to 3’

 I for leading strand

 II RNA primer

 III for lagging strand

 DNA ligase

 DNA gyrase

 DNA is unzipped by unwinding enzyme helicase

 RNA primer join at the replication site

 This primer gives signal to enzyme DNA polymerase to synthesize new DNA strand

• One new DNA strand (Leading strand) is synthesized continuously in the 5’ 3’ direction 

• In contrast, the lagging strand is synthesized discontinuously in fragments (Okazaki fragments)

• RNA primer is digested away by  DNA polymerase I  and synthesizes new DNA to fill in the gap

• DNA ligase joins the discontinuous fragments with covalent bonds 

• DNA polymerase “proofread” each new molecule of DNA and replaces incorrectly paired bases

4. Protein Synthesis

 DNA------- mRNA(transcript)------ protein(translate)

 In Prokaryotes, both transcription and translation occur in the cytoplasm 

 In Eukaryotes: transcription takes place in the nucleus; translation occurs in the cytoplasm. Eukaryotes and Achaeans have regions of genes that code for proteins (exons) and noncoding regions called introns.

5. RNA types

 Three types of RNA participate in protein synthesis: 

◦ Ribosomal RNA (rRNA)

◦ Messenger RNA (mRNA)

◦ Transfer RNA (tRNA)

#microRNA

 Each is synthesized by transcription using single strand of DNA as a template

mRNA

 It carries genetic information from the gene (DNA) out of the nucleus, into the cytoplasm of the cell where it is translated to produce a protein 

 Contains base triplets called codons that constitute the genetic code 

 Attaches to one or more ribosomes

tRNA

 Function is to transfer amino  acids from the  cytoplasm to the  ribosomes for  placement into a  protein molecule 

 Each tRNA molecule consist of  75 to 80  nucleotides folded  back on itself to  form several loops  that are stabilized  by complimentary  base pairing

Structure:  Each tRNA has a three base anti-codon region complimentary to a particular mRNA codon 

 Each tRNA also contains an amino acid binding site, specified by the mRNA codon 

 Amino acid attachment to specific tRNA molecules dictated through action of amino acid activating enzymes and ATP 

 There is a specific tRNA for each of the 20 different amino acids

6. Transcription

 One strand of DNA used as a template to make

a complimentary strand of mRNA

 Promoter/RNA polymerase/termination site/5’ to 3’ 

 Ways in which RNA & DNA differ:

◦ RNA is ss

◦ RNA sugar is ribose

◦ Base pairing-A-U

7. Translation

 Three parts:

◦ Initiation-start codon (AUG)

◦ Elongation-ribosome moves along mRNA

◦ Termination: stop codon reached/polypeptide

released and new protein forms

 rRNA=subunits that form the 70 S ribosomes (protein synthesis occurs here) 

 tRNA=transfers amino acids to ribosomes for protein synthesis)

8. Genetic code

 Standard three letter abbreviation for amino acids known as codons. There are 20 amino acids with 3 base code 

 UAA, UAG and UGA do not code for any amino acids, instead all three code for Stop designating terminator codons 

 AUG codes for Start and the amino acid, methionine (met)

 Therefore, protein synthesis always begins with met

 DNA: triplet code

 mRNA: codon (complimentary to triplet code of DNA)

 tRNA: anticodon (complimentary to codon)

9. Genetic Transfer in Bacteria

 Genetic transfer-results in genetic variation

 Genetic variation-needed for evolution

 Three ways (mechanism) :

◦ Transformation: genes transferred from one bacterium to another as “naked” DNA 

◦ Conjugation: plasmids transferred 1 bacteria to another via a pilus  

◦ Transduction: DNA transferred from 1 bacteria to another by a virus

10. Types and Significance of Gene Transfer 

Define gene transfer.

 Gene transfer: refers to the movement of genetic information between organisms 

 Recombination: The combining of genes (DNA) from two different cells 

 Vertical gene transfer: When genes pass from parents to offspring 

 Horizontal (lateral) gene transfer: Pass genes to other microbes of their same generation

11. Transformation

https://www.youtube.com/watch?v=9U7Kaen2LRA

 A change in an organism’s characteristics because of transfer of genetic information 

 Naked DNA: DNA that has been released from an organism after the cell is lysed and DNA is no longer  incorporated into chromosomes 

 Competence factor is released into the medium and apparently facilitates the entry of DNA into cell

#competence, ability of bacteria to take Naked DNA from the environment & use it, adding DNA lysing agent to the environment stops competence

#S pneumoniae, H influenzae, & Neisseria

12. Conjugation

https://www.youtube.com/watch?v=hm8SZaFmlWg

 Genetic information is transferred from one cell to another 

 Conjugation differs from those other mechanisms in two ways: 

1. It requires contact between donor and recipient cells 

2. It transfers much larger quantities of DNA

 Occasionally whole chromosomes can be transferred

13. The Transfer of F Plasmids

 F plasmids are circular, double-stranded DNA molecules 

 F+ cells contain F (fertility) plasmids and makes and F pilus 

 F- cells lack F plasmids

 F pilus (sex pilus): A conjugation pilus bridge which it attaches to the F- cell

#plasmids

a. F plasmids direct the synthesis of proteins that self-assemble into conjugation pili

b. Resistance plasmids carry genes that provide resistance to various antibiotics

c. Other plasmids direct the synthesis of bacteriocidal proteins called bacteriocins

d. Virulence plasmids carry genes that cause disease

e. Tumor-inducing plasmids cause tumor formation in plants

f. Some plasmids contain genes for catabolic enzymes

14. Transduction  

 A method of transferring genetic material using a bacteriophage 

 Bacteriophage (phage): a virus that can infect bacteria

 Phages: composed of a core of nucleic acid covered by protein coat 

 Phage capable of infecting a bacterium attaches to a receptor site on the cell wall

#generalized (packaging):lytic phage will results in bacterial death

#specialized (excision):lysogenic phage may results in bacterial death, or give the ability to produce exotosin

#Erythogenic toxin, Botulinum toxin, Cholera toxin, Diphtheria toxin, Shiga toxin

15. Genetic Recombination

 The exchange of genes between two DNA molecules to form a new combinations of genes on a  chromosome 

 General

◦ Homologous chromosomes: as part of the sexual cycle of the organism 

◦ Any location

◦ DNA breakage and repair

 Site Specific

◦ Nonhomologous

◦ Viral genomes in bacterial chromosomes

 Replicative

16. Recombinant dna

Restriction enzymes

 recognize and cut, or digest only one particular sequence of nucleotide base in DNA  

 cut this sequence in the same way each time 

 EcoRI, BamHI

17. Transposons

 Transposition: the ability of a genetic sequence to move from one location to another

#moving part of bacterial DNA from chromosome to plasmid, vise versa

#only plasmid can be shared with another bacteria, like vanA gene 

 Transposable element: a mobile genetic sequence

 Insertion sequence: the simplest type of transposable element 

 Transposon（jumping genes）: a transposable element that contains genes for transposition

 stretches of DNA which can insert themselves into new  regions of a chromosome 

The simplest called an insertion element consists of a gene for an enzyme called transposase which required for the insertion  

 can be of benefit to the organism by providing a  mechanism for insertion of  beneficial genes e.g. antibiotics  resistance genes to other non  resistant

18. Methods for inserting foreign DNA 

Transformation

 Electroporation

 Protoplast fusion

 Microinjection

19. Genetic engineering and biotechnology

Lect 8

Gene transfer

1. Determine the types of gene transfer.

 Gene transfer: refers to the movement of genetic information between organisms 

 Recombination: The combining of genes (DNA) from two different cells

 Vertical gene transfer: When genes pass from parents to offspring 

 Horizontal (lateral) gene transfer: Pass genes to other microbes of their same generation

2. Mechanisms of Lateral Gene Transfer in Bacteria 

A. Transformation

 A change in an organism’s characteristics because of transfer of genetic information 

Occur naturally in some species of bacteria, also can be induced in lab

 Naked DNA: DNA that has been released from an organism after the cell is lysed and DNA is no longer incorporated into chromosomes 

 Competence factor is released into the medium and apparently facilitates the entry of DNA into cell 

Electricity, temperature, starvation, cell density

 DNA transport proteins and DNA exonuclease (an enzyme that cuts up DNA) is also needed

The Discovery of Transformation: Griffith’s experiment with pneumococcal infections in mice

#heat shock method

B. Transduction

 A method of transferring genetic material using a bacteriophage through a cytoplasmic bridge

 Bacteriophage (phage): a virus that can infect bacteria 

 Phages: composed of a core of nucleic acid covered by protein coat 

 Phage capable of infecting a bacterium attaches to a receptor site on the cell wall

 Virulent phage: capable of causing infection and the destruction and death of a bacterial cell 

 Temperate phage: ordinarily does not cause a disruptive infection 

 Prophage: DNA that is incorporated into the host bacterium’s DNA 

 Lysogeny: persistence of a prophage without phage replication and destruction of bacterial cell

Bacteriophage Life Cycles: lytic & lysogenic cycle

Specialized Transduction

I. Several lysogenic phages are known to carry out specialized transduction 

II. Phages usually insert at a specific location when they integrate with a chromosome 

III. Lambda phage inserts into the E. coli chromosomes between the gal gene  (galactose) and the bio gene (biotin)

Generalized Transduction 

I. Bacteriophage infection of a host bacterium initiates the lytic cycle 

II. Chromosome is broken into fragments which can be picked up and packaged with phage DNA 

III. Particles are released and infect another bacterial cell 

IV. Host acquires genes that were brought  along (transduced)

C. Conjugation

 Genetic information is transferred from one cell to another 

 Conjugation differs from those other mechanisms in two ways: 

a. It requires contact between donor and recipient cells 

b. It transfers much larger quantities of DNA

 Occasionally whole chromosomes can be transferred

3. The Transfer of F (fertility) Plasmids

 F plasmids are circular, double-stranded DNA molecules 

 F+ cells contain F (fertility) plasmids and makes F pilus 

 F- cells lack F plasmids

 F pilus (sex pilus): A conjugation pilus bridge which it attaches to the F- cell

The Transfer of F’ Plasmids

 DNA incorporated into a chromosome can separate from it and again become an F  plasmid 

 In some cases this separation occurs imprecisely, and a fragment of the  chromosome is carried with the F plasmid 

 Cells containing such plasmids are called F’ strains

4. An F+ x F- mating

I. The cell transfers one strand of DNA from its F  plasmid to the F- cell via  the conjugation bridge 

II. The complementary strands of F plasmid  DNA are synthesized 

III. The recipient cell gets a complete copy of the F  plasmid, and the donor  cell retains a complete  copy

5. High-frequency recombinations (Hfr) 

 Can induce more than 1000X the number of genetic recombinations seen in F+ and F- 

 Hfr strains arise from F+ strains when the F plasmid is incorporated into the bacterial  chromosome at one of several possible sites 

 When an Hfr cell serves as a donor in conjugation, the F plasmid initiates transfer of chromosomal  DNA 

 Initiating segment: part of the F plasmid is transferred along with adjacent chromosomal  genes

6. Plasmids

I. F plasmids direct the synthesis of proteins

that self-assemble into conjugation pili

II. Resistance plasmids carry genes that provide resistance to various antibiotics 

III. Other plasmids direct the synthesis of bacteriocidal proteins called bacteriocins 

IV. Virulence plasmids carry genes that cause disease 

V. Tumor-inducing plasmids cause tumor formation in plants

Lect 9

An Introduction to Taxonomy: The Bacteria

1. Taxonomy

• The science of classification

• Provides an orderly basis for the naming of organisms 

• Places organisms into a category or taxon (plural: taxa) 

• Carolus Linnaeus(1707-1778): 18th century Swedish botanist; the Father of Taxonomy

2. Binomial Nomenclature

• The system used to name all living things

• The first name designates the genus (plural: genera) and its first letter is capitalized 

• The second name is the specific epithet, and it is not capitalized 

• Together the genus and specific epithet identify the species

3. The Meaning of the Names of Some Microorganisms 

• Escherichia coli: Named after Theodore Escherich in 1888; found in the colon 

• Entamoeba histolytica: Ent, intestinal; amoebae, shape/movement; histo, tissue; lytic, lysing or digesting tissue 

• Strain: A subgroup of a species with one or more characteristics that distinguish it from other members of the same species

4. Classification of Human Dog Wolf and a Bacterium

5. Using a Taxonomic Key

• Dichotomous Key: A commonly used key to identify organisms.  

• Has paired statements describing characteristics of organisms.

6. The Five Kingdom Classification

https://microbenotes.com/five-kingdom-system-of-classification-features-and-limitations/

1969, Robert Whittaker

• Fungi

• Plantae

• Animalia

• Protista

• Monera

7.Some Typical Monerans

• Also known as kingdom  prokaryote 

• Prokaryotic organism 

• Lack cell nucleus 

• Lack membrane  bound  organelle 

• Binary fission

• most primitive

Archaeobacteria— Extremophiles able to  exploit the unusual  habitat of a “black  smoker” vent

Mycoplasma

Cyanobacteria

Spirochetes

Gram-negative bacteria

Gram-positive bacteria

Rickettsias (in eukaryotic cell)

8. Some Typical Protists

•Eukaryotic cell 

•Most are unicell 

•Have true membrane enclose nucleus and organelle 

•Live in freshwater, seawater and soil 

•In between

Euglena

Diatom (Pinnularia)

Dinoflagellate (Gonyaulax)

Ciliate (Paramecium)

Sarcodine (Amoeba)

Mastigophoran (Trypanosoma)

Apicomplexan (Plasmodium)

9. Some Typical Fungi

•Multicellular

•Some unicell

•Obtain nutrients from organic matter 

•Structure much simpler than plant 

•Form spore not seed

Club Fungi (Amanita muscaria)

Algae-like Fungi (Pilobolus)

Molds (Penicillium)

Sac Fungi (Morchella)

10. The Three Domains

• A new category even higher than kingdom 

• Archaea

Look like bacteria

Different genes for managing and reading DNA

• Bacteria

Single-celled

No nucleus

• Eukarya

A nucleus

11. Classification

12. Categories of Viruses

a. DNA

Enveloped: herpesvirus

Naked/no enveloped: adenovirus

b. RNA

Enveloped: retrovirus

Naked: picornavirus

Lect 10

Viruses

1. General Characteristics of Viruses

• Viruses are infectious agents that are too small to be seen with a light microscope and that are not cells 

• When they invade susceptible host cells, viruses display some properties of living organisms and so appear to be on borderline between living and nonliving 

• Viruses can replicate, or multiply, only inside a living host cell (obligate intracellular parasites) 

• Viruses contain DNA or RNA but never both

2. Components of Viruses

• Nucleic Acid Core (DNA or RNA)

• Capsid: Surrounding protein coat

• Envelope: Some viruses have this additional surrounding lipid bilayer membrane 

• Virion: A complete virus particle

3. The Components of an Animal Virus

• Viruses use their nucleic acids (genome) to replicate themselves in host cells 

• Capsids also play a key role in the attachment of some viruses. Each capsid is composed of protein subunits called capsomeres. 

• Enveloped viruses have a typical bilayer membrane outside their capsids and acquire their envelope after they are assembled in a host cell and “bud” through host’s membrane 

• Nucleocapsid comprises the viral genome together with the capsid

• Naked: viruses with a nucleocapsid and no envelope 

• Spikes: projections that extend from the viral envelope that may aid in attachment to the host cell 

• Glycoprotein: these surface projections serve to attach virions to specific receptor sites on susceptible host cell surfaces 

• Envelopes help the virus in evading detection by the host’s immune system

4. Viral Shapes

Viral sizes and shapes: Variations in shapes and sizes of viruses compared with a bacterial cell, an animal cell, and a eukaryotic ribosome

• Some viruses are variable in shape, but most have a specific shape that is determined by the capsomeres or envelope 

• Helical capsid: consists of a ribbonlike protein that forms a spiral around the nucleic acid 

• Polyhedral capsid: many-sided, and one of the most common polyhedral capsid shapes is the icosahedron 

• Some viruses have a bullet-shaped capsid and some are spherical

5. Classification of Viruses

• Historically, virologists classified viruses by the type of host/host structures they infected 

• Bacteriophages: infect bacterial cells

• Plant viruses infect plant cells

• Animal viruses are subgrouped by the tissues they attack: 

1. Dermotrophic: if they infect the skin

2. Neurotrophic: if they infect nerve tissue

• Discoveries into biochemical and molecular allowed for viruses to be classified based on: 

1. Type and structure of their nucleic acids

2. Method of replication

3. Host range

4. Other physical and chemical characteristics

• 1966 of the International Committee on Taxonomy of Viruses (ICTV) formed to  establish a single, universal taxonomic  scheme for viruses

Coronaviridae: Coronavirus got their name(corona, Latin for “crown”) 

6. Nucleic Acid Classification

• Major groups of viruses are distinguished first by their nucleic acid content as either DNA or RNA 

• RNA viruses can be single-stranded (ssRNA) or double-stranded (dsRNA) 

• RNA viruses must either carry enzymes or have genes for those enzymes in order to copy RNA genomes after infecting a host cell 

• DNA viruses can also occur in single-stranded or double-stranded form

7. General Properties of RNA Viruses

• Many ssRNA viruses contain positive (+) sense RNA, and during an infection acts like mRNA and can be translated by host’s ribosomes 

• Other ssRNA viruses have negative (-) sense RNA and the RNA acts as a template during transcription to make a complementary (+) sense mRNA  

• Negative (-) sense RNA must carry an RNA polymerase within the virion

Picornaviruses: very small, naked, polyhedral, (+)sense RNA viruses. They include the Enterovirus, Hepatovirus, and Rhinovirus

Retroviruses: are enveloped viruses that have two complete copies of (+) sense RNA. They also contain the enzyme reverse transcriptase, which uses the viral RNA to form a complementary strand of DNA, which is then replicated to form a dsDNA

Rhabdoviruses: Another (-) sense RNA virus group consists of medium-sized, enveloped viruses. The capsid is helical and makes the virus nearly rod or bullet-shaped. Contain an RNA-dependent RNA polymerase that uses the (-) sense strand to form a (+) sense strand that serves as a mRNA and template for synthesis of new viral RNA

Orthomyxoviruses: medium-sized, enveloped, (-) sense that vary in shape from spherical to helical. Their genome is segmented into eight pieces

Reoviruses: have a naked, polyhedral capsid. They are medium-sized dsRNA and replicate in the cytoplasm. Ingestion of only 10 rotavirus particles is sufficient to cause infection and diarrhea

Computer-generated model of a human rhinovirus, cause of the common cold. The colors represent different capsomeres of the capsid

8. General Properties of DNA Viruses

• Like the RNA viruses, the animal DNA viruses are grouped into families according to their DNA organization 

• dsDNA viruses are further separated into families on the basis of: 

1. Shape of DNA (linear or circular)

2. Their capsid shape

3. Presence or absence of an envelope

• Only one family of viruses has ssDNA

Herpesviruses: relatively large, enveloped with linear dsDNA. Widely distributed in nature, and most animals are infected with one or more of the 100 types discovered

Papovaviruses: Named for three related viruses, the papilloma, polyoma, and vacuolating viruses. Small, naked, polyhedral dsDNA that replicate in the nuclei of their host cells. Widely distributed in nature

Parvoviruses: Small, naked, linear ssDNA viruses. Their genetic information is so limited that they must enlist the aid of an unrelated helper virus or a dividing host cell to replicate

9. Emerging Viruses

• Viruses that were previously endemic (low levels of infection in localized areas) or had “crossed species barriers” – that is, expanded their host range to other species 

• Measles are also recurring due to changes in human population densities and travel 

• Yellow fever virus, which has been endemic for more than a century is reemerging and the Dengue fever viruses. Both are carried by mosquitoes

In 2003, an outbreak of “chicken flu” necessitated killing tens of millions of birds

10. Viral Replication

A. Adsorption: the attachment of viruses to host cells 

B. Penetration: entry of virions (or their genome) into host cells 

• Follows quickly after adsorption of the virion to the host’s plasma membrane 

• Unlike bacteriophage, animal viruses do not have a mechanism for injecting their nucleic acid into host cells 

• Most naked viruses enter cell by endocytosis in which virions are captured by pitlike regions on cell surface 

• Uncoating: this process occurs after animal virus enters host cell’s cytoplasm and separates genome from protein coat

Animal virus penetration of host cells: Many naked virions adhere to cell surface and become trapped in pits of cell membrane. Pits invaginate to form separate cytoplasmic vesicles

C. Synthesis: new nucleic acids, capsid proteins, and other viral components 

• Generally, DNA animal viruses replicate their DNA in host cell nucleus with aid of viral enzymes and synthesize their capsid and other proteins in the cytoplasm with aid of host cell enzymes 

• Synthesis in RNA animal viruses takes place in a greater variety of ways than found in DNA viruses 

1. (+) sense RNA acts as mRNA (e.g. picornaviruses)

2. dsRNA (+) sense are transcribed into ssDNA with help of reverse transcriptase (e.g. retrovirus – HIV) 

3. (-) sense RNA make (+) sense RNA which are mRNA (e.g. measles and influenza)

D. Maturation: assembly of newly synthesized viral components into complete virions 

• Maturation: Once an abundance of viral nucleic acid, enzymes, and other proteins have been synthesized, assembly of components into complete virions begins 

E. Release: departure of new virions from host cells

• Release: The budding or new virions through a membrane may or may not destroy the host cell. Adenoviruses bud from host cell in a controlled manner (e.g. shedding) which does not lyse host cells 

• Latent viral infections: this ability is held be all herpesviruses. These dsDNA viruses exhibit a lytic cycle and are activated by a cold, fever, stress or immunosuppression

11. Properties of Bacteriophages

• Can have their genetic information in form of either ds or ss DNA or RNA 

• Relatively simple or complex in nature

• T-even phages (T2, T3 and T6): “T” stands for type 

• Structurally, have a head with genome, tail sheath and plate and tail fibers

Bacteriophage: DNA normally is packaged into the phage head. Osmotic lysis has released DNA from phage, showing the large amount of DNA that must be packaged into a phage

Temperate lambda phage: This virus infects the bacterium Escherichia coli

Replication of an enveloped dsDNA animal virus (e.g. herpesvirus)

Replication of RNA viruses: HIV viruses that are budding from a T-4 lymphocyte

Viral recognition of an animal host cell: Rhinoviruses have “canyons” or depressions, in the capsid that attach to specific membrane proteins on host cell membrane

Viral recognition of an animal host cell: HIV has specific envelope spikes (viral glycoproteins) that attach to a membrane protein receptor on the surface of specific host immune defense cells

14. Culturing of Animal Viruses

• Two discoveries greatly enhanced the usefulness of cell cultures for virologists and scientists 

1. The discovery and use of antibiotics made it possible to prevent bacterial contamination 

2. The discovery of proteolytic enzymes (e.g. trypsin) can free animal cells from surrounding tissues without injuring freed cells 

• Subculturing: the process by which cells from an existing culture are transferred to new containers with fresh nutrient media

Viral culture in eggs: Some viruses, such as influenza viruses, are grown in embryonated chicken eggs

15. Types of Cell Cultures

• Three basic types of cell cultures are widely used in clinical and research virology: 

1. Primary cell cultures: come directly from the animal and if repeatedly subcultured, one cell type will become dominant (cell strain) 

2. Diploid fibroblast strains: Most widely used strain and support growth of a wide range of viruses 

3. Continuous cell lines: consists of cells that will reproduce for an extended number of generations and the most famous is the HeLa cell line

16. The Cytopathic Effect (CPE)

• The visible effect viruses have on cells

• Cells in culture show several common effects, including changes in cell shape and detachment from adjacent cells or culture container 

• CPE can be so distinctive that an experienced virologist can use it to make a preliminary ID of  the infecting virus 

• Syncytia: giant, multinucleate cells caused by fusion of adjacent cells (e.g. paramyxoviruses)

Viral Transformation of Cells:

A. Normal 

B. Transformed

17. Viruslike Agents: Satellites, Viroids, and Prions 

A. Satellites

• Small, single-stranded RNA molecules, which lack genes required for their replication 

• In the presence of a helper virus, they can replicate 

• There are two types:

1. Satellite viruses: most are associated with plant viruses 

2. Satellite nucleic acids (virusoids)

B. Viroids

• 1971: plant pathologist T.O. Diener described a new type of infectious agent when studying potato tuber spindle disease 

• No virions could be detected

• Proposed the concept of a viroid, an infectious RNA particle smaller than a virus

• Viroids have been found to differ from viruses in six ways: 

1. Consists of a single circular RNA molecule of low molecular weight 

2. Exist inside cells, usually inside of nucleoli as particles of RNA without capsids or envelopes 

3. Do not require a helper virus

4. Viroid RNA does not produce proteins

5. Viroid RNA is always copied in the host cell nucleus 

6. Not apparent in infected tissue without use of special techniques to ID nucleotide sequences  in the RNA

Viroids and their effects

A. Viroid particles that cause potato spindle tuber disease

B. The tomato plant on the left is normal; the one of the right is infected with a viroid

Proteineacous Infectious Particles (Prions): Protein structure model of the two forms of prion protein (PrP). The protein helices are represented as spiral ribbons. A: harmless form and B: harmful form

Lect 11

Sterilization and Disinfection

1. Principles of Sterilization and Disinfection

• Sterilization: The killing or removal of all living cells, viable spores, viruses in a material or on an object 

• Sterility: there are no living organisms in or on an object 

• Disinfection: The reduction of the number of pathogenic microorganisms to the point that they pose no danger of disease

• Disinfectant: Typically chemical agents that are applied to inanimate objects 

• Antiseptics: Typically chemical agents that are applied to living tissues

2. The Control of Microbial Growth

• A definite proportion of the organisms die in a given time interval 

• The fewer organisms present, the shorter the time needed to achieve sterility 

• Microorganisms differ in their susceptibility to antimicrobial agents 

• The most susceptible phase for most organisms is the logarithmic growth phase

3. Disinfectant and Antiseptic Response of Staphylococcus aureus

4. The Use of Physical Methods in Control of Microbial Growth

Heat and other physical agents are normally used to control microbial growth and sterilize objects: 

1.Heat

• One of the most popular ways to destroy microbes (flame or boiling) 

• Moist or Dry heat may be used

• Exposure to boiling water for 10 minutes is sufficient to destroy vegetative cells and eukaryotic spores 

• Steam sterilization (autoclaving) is necessary to destroy the bacterial endospore

Sterilization—Hot Air Oven

Small Countertop Autoclave

Large Automatic Hospital Autoclave

Autoclaving

• If water is heated under pressure, its boiling point is elevated, so temperatures above 100°C can be reached

• Pressure: 15 lbs per square inch (psi)

• 15 – 20 minutes at 121°C

• Prions are highly resistant and must be sterilized longer and at higher temperature(134°C for 18 min)

Pasteurization

• A process invented by Pasteur to destroy microbes that caused wine to sour, does not achieve sterility

• Kills pathogens: Salmonella and Mycobacterium

• Milk is pasteurized by heating it to 71.6 °C for at least 15 seconds (flash method)

• Milk is pasteurized by heating it to 62.9 °C for 30 minutes (holding method)

Preservationby Drying

Lyophilization—Manifold Dryer

Lyophilization—Stoppering Tray Dryer

2.Low Temperatures

3.Filtration

• Can be used to sterilize substances that are

destroyed by heat (drugs, serum, vitamins, sucrose)

• To separate viruses from bacteria (manufacture of

vaccines)

• To collect microorganisms from air and water

samples (water quality testing)

4.Radiation

• Ultraviolet radiation (260nm): is quite lethal in destroying microbes but it does not penetrate glass,dirt films, or water

• Ionizing radiation (IR): An excellent sterilizing agent because of its ability to penetrate deep into objects

• IR will destroy bacterial endospores and both prokaryotic and eukaryotic vegetative cells

• X rays and gamma rays are forms of ionizing radiation

• So named because it can dislodge electrons from atoms creating ions

• Damages DNA and produces peroxides (powerful oxidizing agents in cells)

• Deinococcus radiodurans: Able to survive 1000X the amount of radiation that would kill a human(bioremediation of radioactive contaminated sites)

UV Radiation—Serratia marcescens

Microwave Sterilization

Lect 12

MICROBIAL DISEASES AND CONTROL

1. TERMINOLOGY

 infection

◦ growth and multiplication of parasite on or within host

 communicable disease

- disease can be transmitted from one host to another

 infectious disease

◦ disease resulting from infection

 pathogen

◦ any parasitic organism that causes infectious disease

◦ primary pathogen – causes disease by direct interaction with host

◦ opportunistic pathogen – causes disease only under certain

circumstances

 pathogenicity

◦ ability of parasite to cause disease

 virulence

◦ degree or intensity of pathogenicity

2. Pathogenesis of Viral Diseases

 enter host

 contact and enter susceptible cells

 replicate within cells

 spread to adjacent cells

 cause cellular injury

 engender host immune response

 be cleared from host, establish persistent infection, or kill host

3. Entry, Contact, and Primary Replication

 entry

◦ via body surface

◦ via needle, blood transfusions, and organ transplants

◦ via insect vectors

 primary replication

◦ some replicate at site of entry

◦ others spread to distant sites and then replicate

4. Pathogenesis of Bacterial Diseases

 maintain a reservoir

◦ place to live before and after causing infection

 be transported to host

 adhere to, colonize, and/or invade host

 multiply or complete life cycles on or in host

 initially evade host defenses

 damage host

 leave host and enter new host

5. Transport of the Bacterial Pathogen to the Host

 direct contact

◦ e.g., coughing, sneezing, body contact

 indirect contact

◦ vehicles (e.g., soil, water, food)

◦ vectors – living organisms that transmit pathogen

◦ fomites – inanimate objects that harbor and transmit pathogens (e.g doorknob toilet seat, catheter, etc.)

6. Attachment and Colonization by the Bacterial Pathogen

 adherence

◦ mediated by special molecules or structures called adhesins

 colonization

◦ establishment of a site of microbial reproduction on or within host

7. Invasion of the Bacterial Pathogen

 can be active penetration of host’s mucous membranes or epithelium

 can be passive penetration (e.g., skin lesions, insect bites, wounds)

 once below mucous membrane, bacterium can spread to deeper tissues - involves production of specific products and/or enzymes that promote spreading

8. Growth and Multiplication of the Bacterial Pathogen

 occurs when pathogen finds appropriate environment within host

 some bacteria invade specific cells

 some actively growth in blood plasma

 bacteremia – presence of viable bacteria in blood

 septicemia – presence of bacteria or their toxins in bloodstream

Virulence factor

9. Exotoxins and Endotoxin

10. Microbial Mechanisms for Escaping Host Defenses

1. Evasion of Host Defenses by Viruses

 mutations that change antigenic sites or alter expression of antigens

 infection of immune system cells, diminishing their function

 infection of tissues with few MHC (major histocompatibility complex) molecules

 production of proteins that inhibit MHC

 production of free antigens that bind neutralizing antibodies

2. Evasion of Host Defenses by Bacteria

 have mechanisms to resist complement system, phagocytosis, and specific immune responses.

1. Evading the complement system

2. Resisting phagocytosis

3. Survival inside phagocytic cells

4. Evading specific immune response molecule function

11. Epidemiology

 science that evaluates occurrence, determinants, distribution, and control of health and disease in a defined human population

 endemic disease - maintains a relatively steady low-level frequency at a moderately regular interval--Denggi AEDES

 hyperendemic diseases - gradually increase in occurrence frequency above endemic level but not to epidemic level

 Epidemic - sudden increase in frequency above expected number (index case – first case in an epidemic)

 Pandemic - increase in disease occurrence within large population over wide region (usually worldwide)--Covid-19

12. The Epidemiologist’s Tools

 statistics

◦ mathematics dealing with collection, organization, and interpretation of numerical data

 three important statistical measures of disease frequency

◦ morbidity rate

- an incidence rate

- number of new cases in a specific time period per unit of population

# new cases during a specific time

# individuals in population

◦ prevalence rate

- total number of individuals infected at any one time

- depends both on incidence rate and duration of illness

◦ mortality rate

- number of deaths from a disease per number of cases of the disease

# deaths due to given disease

size of total population with disease

13. Infectious Disease Epidemiology

 tries to determine:

◦ causative agent

◦ source and/or reservoir of disease agent

◦ mechanism of transmission

◦ host and environmental factors that facilitate development of disease within a defined population

◦ best control measures

14. Course of infectious disease IPPC

 incubation period

◦ period after pathogen entry but before signs and symptoms appear

 prodromal stage

◦ onset of signs and symptoms

◦ not clear enough for diagnosis

 period of illness

◦ disease is most severe and has characteristic signs and symptoms

 convalescence

◦ signs and symptoms begin to disappear

15. The 5 stages of an infectious disease.

1. Incubation Period. Time between infection and the appearance of signs and symptoms. Infected person is not aware of the presences of the infectious agent, they can spread the disease.孵化

2. Prodromal Phase. Short period during which nonspecific symptoms sometimes appear. Individuals are contagious and can spread the disease to others.传染

3. Invasive Phase. Individual experiences the typical signs and symptoms of the disease. Can include fever, nausea, headache, rash. Signs and symptoms reach their greatest intensity is known as acme. During acme, pathogens invade and damage tissues.症状

4. Decline Phase. Symptoms begin to subside, and the disease enters the decline phase. The decline phase is the period of illness during which the host defenses and the effects of treatment finally overcome the pathogen.痊愈

5. Convalescence Period. Tissues are repaired, healing takes place and the body regains strength and recovers. Individuals no longer have disease symptoms免疫

16. What Pathogen Caused the Disease?

 clinical microbiology lab

◦ plays important role in isolation and identification of pathogen

 communicable disease

◦ can be transmitted from one host to another

17. What was the Source and/or Reservoir of the Pathogen?

 source来源

◦ location from which pathogen is transmitted to host

 period of infectivity

◦ time during which source is infectious or is disseminating the organism

 Reservoir

◦ site or natural environmental location in which pathogen is normally found

◦ sometimes functions as source of pathogen

Human sources/reservoirs

 carrier携带

◦ infected host

 active carrier

◦ has overt clinical case of disease

 convalescent carrier

◦ has recovered from disease but continues to harbor large numbers of pathogen

 healthy carrier

◦ harbors pathogen but is not ill

 chronic carriers

◦ convalescent, healthy, and incubatory carriers that harbor pathogen for long time

Animal reservoirs

 numerous diseases are zoonoses. (the process whereby an infectious disease is transmitted between species).

 transmission to human can be direct or indirect

 vectors媒介

◦ organisms that spread disease from one host to another

18. How Was the Pathogen Transmitted?传播

 four main routes

• airborne空气

Airborne Transmission

 pathogen suspended in air and travels  1 meter

 droplet nuclei

◦ small particles (1-4 m diameter)

◦ can remain airborne for long time

◦ can travel long distances

◦ usually propelled from respiratory tract of source organisms by sneezing, coughing, or vocalization

 dust particles also important route of airborne transmission

• contact接触

Contact Transmission

 coming together or touching of source/reservoir and host

 direct contact (person-to-person)

◦ physical interaction between source/reservoir and host

◦ e.g., kissing, touching, and sexual contact

 indirect contact

◦ involves an intermediate (usually inanimate)

◦ e.g., eating utensils餐具, bedding家具

 droplet spread

◦ large particles (>5 m) that travel < 1 meter

• vehicle非生物/物体/食物/水源

Vehicle Transmission

 vehicles

◦ inanimate materials or objects involved in pathogen transmission

 common vehicle transmission

◦ single vehicle spreads pathogen to multiple hosts

◦ e.g., water and food

 fomites

◦ common vehicles such as surgical instruments, bedding, and eating utensils

• vector-borne媒介

Vector-Borne Transmission

 external (mechanical) transmission身上

◦ passive carriage of pathogen on body of vector

◦ no growth of pathogen during transmission

 internal transmission体内

◦ carried within vector

◦ harborage transmission – pathogen does not undergo changes within vector

◦ biologic transmission – pathogen undergoes changes within vector

19. Nosocomial Infections医院感染

 result from pathogens that develop within a hospital or other clinical care facility and are acquired by patients while they are in the facility

 5-10% of all hospital patients acquire a nosocomial infection

 develop during a stay at a medical facility

 People already sick more susceptible high density > spread contact with medical instruments.

20. Control, Prevention, and Surveillance监视/观察

 proper training of personnel in basic infection control measures

◦ e.g., handling of surgical wounds and hand washing

 monitoring of patient for signs and symptoms of nosocomial infection

21. The Hospital Epidemiologist

 individual responsible for developing and implementing policies to monitor and control infections and communicable diseases

 reports to infection control committee or similar group

22. Drug Susceptibility Testing

 testing for susceptibility to antimicrobial agents

 can be used to identify microbe

 particularly useful for determining proper therapy

 determined by:

◦ dilution susceptibility tests

◦ disk-diffusion tests (Kirby-Bauer method)

◦ E test

23. Chemotherapeutic agents

 chemical agents used to treat disease

 destroy pathogenic microbes or inhibit their growth within host

 most are antibiotics

◦ microbial products or their derivatives that kill susceptible易受影响 microbes or inhibit their growth

24. General Characterisitics of Antimicrobial Drugs

 selective toxicity

◦ ability of drug to kill or inhibit pathogen while damaging host as little as possible

 therapeutic dose剂量

◦ drug level required for clinical treatment

 toxic dose

◦ drug level at which drug becomes too toxic for patient (i.e., produces side effects)

 therapeutic治疗 index

◦ ratio of toxic dose to therapeutic dose

25. Determining the Level of Antimicrobial Activity

 effectiveness expressed in two ways:

◦ minimal inhibitory抑制 concentration (MIC)

- lowest concentration of drug that inhibits growth of pathogen

◦ minimal lethal concentration (MLC)

- lowest concentration of drug that kills pathogen

26. Factors Influencing the Effectiveness of Antimicrobial Drugs

 ability of drug to reach site of infection

- depends in part on mode of administration (oral, parenteral, topical)

 susceptibility of pathogen to drug

 ability of drug to reach concentrations in body that exceed MIC of pathogen

- amount administered

- route of administration

- speed of uptake

- rate of clearance (elimination) from body

27. Preventing emergence of drug resistance

 use drugs only when necessary

 possible future solutions

◦ continued development of new drugs

◦ use of bacteriophages to treat bacterial disease

SUMMARY

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