prokaryotes:

no nucleus or membrane bound organelles

less than 5 microns in size

70s ribosomes (eukaryotes 80s except in organelles which have 70s)

nucleic acid is a single circular molecule

replicate by binary fission

thin peptidoglycan layer

have outer membrane and periplasmic space (pps)

porins transport hydrophilic molecules in/out of pps

LPS and lipid A major surface antigen

stain red or pink: lose crystal violet and get safranin red

thick peptidoglycan

teichoic acids - major surface antigen

stain purple: retain crystal violet

1) crystal violet

2) gram's iodine - form complex in cells that have crystal violet

3) decolorizer (ex alcohol)

4) safranin red

Lipid A - endotoxin activity

core polysaccharide: link Lipid A to terminal PS, maintain cell wall integrity

terminal PS: repeating sugar subunits; O antigen

no sterols (few exceptions: mycoplasma)

no organelles: cell membrane takes over these processes

(respiration, making macromolecules)

AKA glycocalyx, exopolysaccharide, slime layer

hydophilic, gel like simple polysaccharide covering

aid in identification

avoid phagocytic recognition

weak immunogen

can allow growth of microbial communities

hair-like filamentous structures that extend outward through the capsular layer

anchor bacteria to solid surfaces

outer part: single repeating protein subunit

Additional fimbrial proteins are required for membrane transport, anchoring to the cell wall, filament assembly

filamentous structures that extend outward from the cell to function in motility

single repeating protein subunit, flagellin

Additional proteins are involved in hook and basal body structures which anchor the flagellum to the cell wall and generate motion

made by bacillus and clostridium

extreme resistance: dipicolinic acid, high levels of
calcium in the core wall

Parts:

core: contains components of live cell

inner membrane: semi crystalline, becomes fluid after germination. surrounded by thin PG layer

cortex: modified PG, special glycosidic bonds resist lysozyme, no teichoic acids or carbs

coat: impermeable to enzymes

exosporium: thin, complex membrane on outside

takes several minutes

end dormancy: physical damage to the exosporium

germination is initiated by loss of dipicolinic
acid and calcium and uptake of water.

Outgrowth through the spore coat and cell division: when nutrients are present

cell dies and releases an endospore

takes 8-10 hours

cells rapidly reproduce

produce metabolic enzymes

find growth rate by measuring colonies, or possibly spectrophotometry of samples, over time

"population doubling time"

if new nutrients are added and toxic products are removed

in a steady-state, bacteria can, theoretically,
be maintained in logarithmic phase forever.

A laboratory device used to obtain a steady-state of
bacterial growth is called a chemostat.

Selective-enrichment and selective-differential media both select for a particular type of bacteria. 

Selective-differential media differs in that it also contains some substance, such as a pH indicator, to assist in determining the identity of the bacteria.

Obligate aerobes

microaerophilic bacteria

capnophilic bacteria: carbon dioxide also must be high

aerotolerant

facultative anaerobes

obligate anaerobes

Superoxide, singlet oxygen, peroxides

found: where metabolism is occuring; especially high in phagolysosomes

Bacteria use enzymes (oxidase, catalase, superoxide dismutase) to convert them into less dangerous forms.

Refrigeration, freezing, treating with acid or salt, drying.  Heating, radiation, and preservatives are intended to kill bacteria.

Procedures (hand-washing)

equipment (gloves)

facilities (double sets of doors, autoclaves, security)

virulence and therapy groups:

1 – unlikely to cause disease. 

2 – associated with non-serious disease, treatment available. 

3 – associated with serious disease, treatment sometimes available. 

4 – likely to cause serious disease, treatment unavailable or disease difficult to treat. 

risk to individual and community: 

1 – low risk.  

2 – moderate individual risk. 

3 – high individual risk, low community risk. 

4 – high individual and community risk.

likelihood  (%) of true positives found by your test. 

likelihood (%) of true negatives found by your test. 

represents how likely a positive result is to be a true positive

varies with prevalence of the disease in the population

represents how likely a negative result is to be a true negative. 

Proper test selection

proper sample collection

labeling

storage

transport

Direct – Gross or microscopic

   Gram stain and acid fast stain

 Serology / fluorescent antibody

molecular analysis: PCR and DNA restriction comparisons.

Serology (ELISA testing)

skin hypersensitivity testing

lymphocyte proliferation 

cytokine production assays

  Limitations:

you have to know what you’re testing for. 

not cheap enough to test for everything at once

some combined tests are available (e.g. Snap Tests for lyme, ehrlichia, and heartworm).

naturally produced by fungi and bacteria

purpose: "secondary metabolites" - not certain

vestiges of normal metabolism

could be used for signalling

1st order rate kinetics, i.e. one molecule of drug is consumed by one target molecule.

maintaining an optimal proportion of drug to target is important for treatment.

Cell wall – beta lactams (penicillin)

                Cell membrane – polymixin

                Ribosomal action – tetracyclines (doxycycline)

       Nucleic Acid:

sulfonamides (sulfamethazine) - folic acid synth

metronidazole - DNA structure

Quinolones (enrofloxacin) - DNA gyrase

Diffusion: diffuse drug through agar to form a gradient of drug concentrations

ex: disc diffusion

Dilution: contain uniform concentrations of drug in a broth or agar medium

ex: microbroth diluton

diameter of zone of inhibition

compare to standard values for organism to classify as

Susceptible, Intermediate or Resistant

visible growth

MIC is reported as lowest concentration where all growth is inhibited

composition of medium

ions / pH

depth and volume

concentration of drug

disk size and material

incubation time and conditions

preparation of inoculum, concentration

organism used

criteria for endpoints

high likelihood of therapeutic success

MIC or zone diameter values compared to published “breakpoint” guidelines

    MICs from large,
    geographically diverse populations of the bacterium
    isolated in the field

    pharmacokinetic and pharmacodynamic properties of the drug in the host

    efficacy of the drug in clinical trials at standard dosage and dosing interval in the target
    infected host

inhibitory concentrations assume the drug is in the plasma

the lethality (in animal studies) divided by the effective dose

values are found for 50% of a representative population (LD50 and ED50)

high therapeutic index: drugs that are able to target a part of the bacteria not present or important in the host

ex: cell wall synthesis / beta lactams

kills bacteria

ex: beta lactams (must have active growth) cephalosporins

slows the growth of bacteria without killing

ex: tetracyclines, sulfonamides

small, circular peices of DNA

do not code essential functions or products

Conjugative plasmids are self-transmissible from one bacterium to another in the process called conjugation

Some non-conjugative plasmids can be mobilized by conjugative plasmids, others cannot

plamids are replicated when the cell divides, can be integrated in the chromosome 

Recognition

Injection

Transcription of phage DNA

replication & protein synthesis

phage assembly

lysis and release
Lysogenic cycle: viral DNA is integrated into the bacterial chromosome and coexists with it; no new viruses are made but DNA is replicated when host reproduces
stress brings on the lytic cycle

small, extracellular DNA fragments

free DNA is rapidly degraded in extracellular environments and DNA is a large macromolecule that does not normally cross cell membranes.

competent cells are able to bind the DNA, translocate accross membrane and integrate into its chromosome

within one species

bacteria die and others are able to pick up their DNA to get that function

occurs in a limited number of pathogens; mostly within same species

moderate gene capacity

a DNA fragment is assembled in a bacteriophage nucleocapsid during lytic infection

Transduction occurs in different bacterial species but is limited by host range of the transducing phage

moderate to large gene capacity

unidirectional plasmid transfer from a suitable donor to a recipient bacterial cell

encodes sex pilus so the recipient can go on to transfer it again

site-specific recombination systems that recognize sites on 2 DNAs and promote the insertion of new DNA.

The most common integrons possess an antimicrobial resistance gene.

Integrons are important factors in multiple antimicrobial resistance in Gram-negative bacteria.

may move by excision from one location and insertion into another or by replication at one site and insertion of a new copy elsewhere.

sometimes referred to as jumping genes, can move large distances between DNA regions within a cell, eg. between plasmids and chromosome.

Many transposons possess genes for other traits such as antimicrobial resistance.

insertion may result in inactivation of genes in the target DNA region

genetic elements that are capable of moving, in a random fashion,from one place in a DNA molecule to another through site specific recombination

Each has terminal invert repeat sequences that encode enzymes for excision (transposase) and insertion (recombinase) and is flanked by direct repeats of the target sequence following insertion.

Insertion sequences are always excised from one region of DNA and inserted into another.

Insertion sequences do not move great distances and do not usually carry selectable genes.

1. Prudent drug use

restrict extra label use, alternative therapy

2.Pathogen Control

hygeine, vaccination, food safety, understanding resistance methods

3. Policy

education, resisitance monitoring

 1) microbe must be associated with sick animals and not with healthy ones. 

2) grow a pure culture of the microbe. 

3) Infect a new host with the microbe and see disease take hold. 

4) isolate the same microbe again from the new host.  

  Limitations:

some microbes are opportunistic (so they appear in healthy as well as sick hosts),

not all microbes can be cultured,

in culture the microbes could lose their virulence.  

multi-species disease – a disease caused by the interaction of two or more parasitic microbe species.

host: species, age, breed, sex, genetics, physiology, immune

 pathogen: virulence, stability, route, dose

 environment: stress, nutrition, trauma, immunosuppression, metabolic dysfunction, intercurrent disease, cilmate

1. enable a bacterium to reach a site of entry (bacterial motility and chemotaxis)

2. hold bacteria in place at the site of entry (bacterial adhesins – fimbriae, non-fimbrial

surface proteins, biofilm-forming exopolysaccharides)

3. support sufficient growth at the site of entry (preferred substrates, iron sequestering

components – siderophores, lactoferrin/transferring/heme receptors)

Colonization can involve complex relationships with other microorganisms.

possession of iron sequestering components (meet nutrient requirements)

avoiding destruction by host phagocytic cells and serum proteins:

1. evade phagocytosis 

2. destroy or inactivate phagocytes 

3. protect bacteria from phagocytic killing by escape from phagocytic vacuoles, prevention of

lysosomal fusion to phagosomes or resistance to hydrolysis in phagolysosomes. 

Toxins: act directly with mechanical or physiological alterations in cells 

act indirectly by triggering exaggerated host inflammatory/immunological responses

that cause tissue damage

1) a requirement for unfolding through enzymatic “nicking” to activate toxin 

2) functional domains (binding and catalytic activity) that are physically separable either as different

proteins or different termini of a single protein.