Intro Micro Lecture 1,2,3

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B. The number of deaths from infectious diseases is increasing, even in developed countries due to HIV/AIDS and other factors, like:

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1. emerging infectious agents
2. increased number of elderly people
3. increased number of immunocompromised people
4. hospitals and invasive procedures and devices; nosocomial infections
5. more foods prepared outside of the home

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What are examples of infectious diseases that cause morbidity + mortality in economically challenged countries?

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tuberculosis, HIV/AIDS, infantile diarrhea due to rotavirus, cholera, bacterial meningitis, certain parasitic diseases, like malaria, and other “neglected tropical diseases” (e.g. onchoceriasis, aka “river blindness”, dracunculiasis, aka “guinea worm disease”, schistosomiasis)

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Name the contributions of the following historians
1) Antony van Leeuwenhoek
2) Louis Joblot
3) Lazzaro Sallanzani
4) Ferdinand Cohn
5) Robert Koch

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A. Antony van Leeuwenhoek (ca. 1675) and the theory of replicating microbes vs. spontaneous generation.

B. Louis Joblot (ca. 1718) vs. John Needham (ca. 1749) and the theory of sterilization.

C. Lazzaro Spallanzani (ca. 1170) and Louis Pasteur (ca. 1862) sterilization by autoclaving (115-1200C).

D. Ferdinand Cohn (1876) and the discovery of spores.

E. Robert Koch (1877) described methods for heat-fixing bacteria to slides for staining and microscopic examination, and for isolating pure cultures of bacteria on solid media.

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1) Commensals
2) Commensal microorganisms.

Where do they reside?

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1) two organisms living in a close relationship in which one organism benefits from the relationship and the other one neither benefits nor is harmed.
2)make up the normal flora of the human body, are generally harmless and actually protect the host from invasion by pathogenic microorganisms

3) reside on skin, in the oral cavity, and on mucous membranes of the upper respiratory tract, intestines, and vagina

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Opportunistic pathogens

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part of the normal flora in healthy people and they do not cause disease under these conditions. However, they can cause disease when they become “dislocated” or introduced into new sites in the body (ex. blood, CSF). These organisms can also cause disease in patients whose immune system is compromised or who are in generally poor health (ex. malnourished, ill due to other causes, etc.).

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1) pathogens
2) strict pathogens
3) colonizing microorganisms
4) infectious/contagious

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1) microorganisms that are adapted to defeating the natural host defenses and invading the tissues, a process called infection
2) microorganisms whose presence is always associated with disease.
3) those that appear as new organisms in a host, but they are not existing members of the normal flora or the cause of symptoms. They may be transient (present for short periods of time (hours to months) in some people) or become permanent members of the normal flora (present in most people most of the time).
4) diseases that are readily transmitted from person to person

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1) vertical transmission
2) horizontal transmission

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1) transmission from mom to offspring in utero or perinatally
2) transmission from person to person

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Germ Theory of Disease

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The transmissible nature of diseases led to identification of microbes as the
causative agents. The germ theory states that microorganisms are
responsible for causing certain human diseases

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Describe Koch's postulates

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2. Koch’s Postulates must be fulfilled to prove that a particular microorganism is the cause of a particular disease. The microbe must:

a. be found in the body in all cases of the disease.

b. be isolated from a case of the disease and grown in a series of pure cultures in vitro.

c. reproduce the disease when a pure culture is inoculated into a susceptible animal.
d. be recovered from the inoculated animal.

e. It may not be possible to fulfill Koch’s postulates in all cases in which a microorganism is the causative agent of disease. Can you think of reasons why?

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Name how the following microorganisms were discovered, and what did they cause?

1) Fungi
2) protozoa + helminths
3) viruses
4) prions

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1) David Gruby (1841-1845) identified yeast infections as the cause of thrush and
fungi as the cause of ringworm
2) Leeuwenhoek observed protozoa, most likely Giardia lamblia in his own feces, in
1676.

2. Most of the protozoa and helminths (worms) that infect humans were described in
the 19th century
3) Much more difficult to demonstrate as causative agents of disease.
a. Invisible by light microscopy (need an electron microscope); filterable.

b. Obligate intracellular parasites (need cell culture or animals to propagate
4) Novel infectious agent composed solely of protein. Like the idea of bacteria and
viruses once did, these agents challenge our fundamental ideas of infectious agents

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Bacteria are given 2 names, what do the first and second name correspond to?

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1. first name- genus name
2. last name - refers to species (not capitalized)

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How do you make a presumptive identification?

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Grow bacteria on agar plates to see their characteristic colonial morphologies.

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1) alpha- hemolytic
2) Beta hemolytic
3) non-hemolytic

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1) An incomplete hemolysis of red blood cells results in a greenish-brown colored zone surrounding the colony.
2) Complete hemolysis of red blood cells results in a clear/colorless zone surrounding the colony.
3) No hemolysins are produced and no lysis of red blood cells occurs.

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Name reasons why the following bacteria cannot be seen in the Gram stain.

1) Mycobacteria (M. tuberculosis)
2) Treponema pallidum
3) mycoplasma pneumoniae
4) legionella pneumoniae
5) chlamydiae
6) rickettsiae

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1) too much lipid in cell wall so dye cannot penetrate (alt approach is to use acid fast stain)
2) too thin to see (dark-filed microscopy)
3) no cell wall; very small. No alternative methods
4) poor uptake of red counterstain (alt method: prolong time of counterstain)
5) intracellular; very small (alt method - look for inclusion bodies in cytoplsm, but insensitive)
6) intracellular; very small (alternative approach: Giemsa, other tissue stain)

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Name 3 types of cell arrangements and what their orientation is determined by

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types: clusters, chains, pairs

Determined by orientation and degree of attachment of the cells during cell division.

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What is biotyping based on?

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Based on the presence or absence of specific BIOCHEMICAL markers, like:
a. ability to ferment specific sugars.
b. production of specific end products.
c.relationship to oxygen.

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What is serotyping based on?

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based on detection of unique antigens using specific ANTIBODIES
advantages: fast, used for organisms that can't be identified by biotyping, can be used for organisms difficult or impossible to grow.

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What are antibiograms?
2. phage typing?

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1. tests for susceptibility to different antibiotics
2. tests for patterns of susceptibility to infection by bacteriophages

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Name 3 types of classifications that can be done through genotyping.

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1. G + C content
2. DNA hybridization
3. nucleic acid sequence analysis (PCR)

Front 20

Name 3 components of the bacterial cell wall.

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1) peptidoglycan
2) outer membrane of gram-negative bacteria
3) surface fibers in some gram-positive bacteria

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Describe the chemical components of peptidoglycan, the outer membrane of gram neg, and the surface fibers of gram + bacteria

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1) sugar backbone w/ peptide side chains that are cross linked
2) lipid A + polysaccharide
3) teichoic acid, lipteichoic acid

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Describe the functions of the 3 compoenents in the cell wall.

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1) peptidoglycan: Provides rigid support and shape, protects against osmotic pressure; site of action of penicillins & cephalosporins; is degraded by lysozyme.
2) lipid A- Toxic component of endotoxin. polysaccharide - Major surface antigen used frequently in lab diagnosis.
3) LPS-like (fibers in Gram + bacteria)

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Name the chemical composition and function of the cytoplasmic membrane

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1) lipoprotein bilayer w/o sterols. fcn: site of oxidative and transport enzymes

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chemical comp and function of ribosomes in bacteria

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1) RNA, protein 50S and 30S (70s)
Protein synthesis; site of action of aminoglycosides, erythromycin, tetracyclines & chloramphenicol.

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Chemical composition and function of nucleoid and periplasm of gram neg bacteria

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1) DNA - genetic material
2) space between plasma + outer membrane. Contains many hydrolytic enzymes, including
β-lactamases.

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1) Capsule
2) pilus/fimbriae
3) flagellum

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1) polysaccharide - protects against phagocytosis
2) glycoprotein ; 2 types - mediates attachment to cell surfaces; sex pilus mdiates attachment of 2 bacteria during conjugation + transfer of genetic material
3) protein; motility

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4) spore
5) plasmid
6) granule
7) glycocalyx

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4) keratin-like coat; dipicolinic acid. provides resistance to dehyd, heat, and chem; germination gives rise to single cell
5) DNA; contains a variety of genes for antibiotic resistance and toxins
6) glycogen, lipids, polyphosphates; site of nutrients in cytoplasm
7) mediates adherence to surfaces

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Describe differences in cell walls of gram positive and gram negative cells

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1) peptidoglycan is thicker and multilayered in gram + cells; thinner and single layer in -.
2) teichoic acids are only in some species of gram +
3) LPS and periplasmic space are only in Gram -

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What does mycobacteria species have in the wall that are not in Gram + and Gram - bacteria?

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has high concentration of lipids called mycolic acids that can't be gram stained.

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What is peptidoglycan?

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Carbohydrate backbone composed of alternating N-acetylmuramic acid
and N-acetylglucosamine molecules. A tetrapeptide consisting of both D-and L-amino acids is attached to each muramic acid molecule. The composition of the tetrapeptide differs among bacteria

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What amino acids are only present in bacterial cells?

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Diaminopimelic acid, and D-Ala is involved in cross links bet. tetrapeptides + penicillin.

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How does lysozyme work?

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d. Lysozyme cleaves the peptidoglycan backbone by breaking glycosyl bonds. Lysozyme is present in human tears, saliva and mucous and is a natural defense to bacterial infection.
i. Lysozyme treatment weakens the cell wall, so that bacterial cells will swell and rupture, unless they are in a solution with the same
osmotic pressure as inside the bacterial cell. These spherical
forms, called protoplasts (derived from Gram-positive bacteria) or spheroplasts (derived from Gram-negative bacteria), survive and divide with the cytoplasmic membrane and little or no peptidoglycan (in an isotonic solution).

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What are the 3 units in LPS?

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i. Lipid A—a phospholipid responsible for the toxic effects.
ii. A core polysaccharide of 5 sugars linked to lipid A.
iii. An outer polysaccharide consisting of up to 25 repeating units of 3-5 sugars; the somatic or O antigen of several Gram-negative bacteria used for clinical identification.

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Teichoic acid?

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Water-soluble polymers (fiber-like) of glycerol or ribitol phosphate covalently linked to peptidoglycan in the outer layer of Gram-positive cell wall and extending from it. Fibers penetrating the peptidoglycan layer and covalently linked to lipid in the cytoplasmic membrane are called lipoteichoic acid.

b. Induces septic shock caused by certain Gram-positive bacteria by activating the same pathways as LPS.

c. Mediates attachment of Staphylococcus aureus to mucosal cells

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Describe the outer cell membrane of gram - bacteria cells

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1. asymmetric bilayer structure; inner leaflet similar to other phospholipid memb., outer leaflet has LPS
2. has PORINS - pores for diffusion of small hydrophillic molecules
3. invovled in signal transduction (quorum sensing - comm among bact)
4. polymyxin and other antibiotics can disrupt it.

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what are the components of the cytoplasmic cell membrane in gram - bacteria?

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1. Located just inside the peptidoglycan layer.

2. Phospholipid bilayer similar to eukaryotic membranes, but does not contain sterols (except Mycoplasma species).

3. Functions:
a. active transport of molecules into cell
b. energy generation by oxidative phosphorylation
c.synthesis of cell wall precursors
d.secretion of enzymes and toxins
e.signal transduction

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4 reasons the capsule is important

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i. It is a virulence factor; inhibits phagocytosis.
ii. Antibodies vs. capsular polysaccharides used to identify bacteria
a. Quellung reaction used in clinical labs—when homologous antibody present, capsule swells.

iii. Capsular polysaccharides elicit protective antibodies as antigens
in certain vaccines; Ex. Capsular polysaccharides from 23
types of Streptococcus pneumoniae are in the current vaccine.

iv. It may play a role in bacterial adherence to human tissues and in formation of biofilms

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What's the role of the flagella?

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Long, filamentous appendages that propel bacteria towards
nutrients and other attractants (chemotaxis).
b. Composed of multiple copies of a single protein, flagellin organized in intertwined chains.
c. Proton motive force provided by ATP provides energy for movement.
d. Present in characteristic numbers (1 or many) and arrangements (at the end or all over the bacterial surface).
e. Most common in rods, not cocci.
f. May play a role in pathogenesis; Ex. E. coli and Proteus vulgaris are common causes of urinary tract infections (UTIs), perhaps because their flagella move them up the urethra into the bladder.
g. Antibodies vs. flagellar proteins are used to identify some bacterial species; Ex. Salmonella species.

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Role of pili (fimbriae)

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a. Hair-like filaments extending from cell surface.

b. Shorter, straighter than flagella.

c. Composed of the protein, pilin, organized in helical strands.

d. Found mainly on Gram-negative organisms.

e. Mediate attachment of bacteria to specific receptors on human cells; via adhesins; can be considered a virulence factor.

f. Sex pili function during conjugation.

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What's glycocalyx?

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Polysaccharide coating secreted by many bacteria that mediates
adherence to skin, heart valves, catheters, etc. even teeth (basis of
plaque and cavity formation); i.e. formation of biofilms. Similar to a
capsule, but more loosely constructed.

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Spores?

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a. Highly resistant structures formed intracellularly by certain bacteria (Gram-positive bacilli: Bacillus and Clostridium) when nutrients are scarce.

b. Composed of bacterial DNA, small amount of cytoplasm, cell membrane, peptidoglycan, a little water, and a thick, keratin-like coat.

c. The outer coat provides remarkable resistance to heat, dehydration, radiation and chemicals; likely due to dipicolonic acid, a calcium ion chelator found only in spores.

d. Have no metabolic activity and can remain dormant for years.

e. When water and nutrients become available, spore may germinate and give rise to a single bacterial cell.