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(Added new sections on viri, fungi and others)
m (=rna viruses: fixed layout)
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**papoviridae and polymaviruses
 
**papoviridae and polymaviruses
  
===rna viruses==
+
===rna viruses===
 
*learn mostly single stranded ones
 
*learn mostly single stranded ones
 
*positive (+) stranded rna - genomes of the same polarity as mRNA  
 
*positive (+) stranded rna - genomes of the same polarity as mRNA  

Revision as of 03:09, 11 March 2008

Contents

Medical Microbiology

Study of microscopic organisms includes

  • Bacteriology Bacteria simplest smallest single celled free living organisms
  • Virology viruses - non cellular parasitic, not living organisms
  • Mycology fungi - microscopic (molds and yeast), macroscopic (mushrooms and puff balls), unicellular and multicellular
  • Protozoology - protozoa, single celled
  • Phycology/Algology - algae - simple aquatic organisms ie seaweeds

Koch's postulates

  1. The same microbe is always associated witha specific disease
  2. this microbe can be recovered and grown in pure culture
  3. The pure culture must cause disease in an experimental animal
  4. The original microbe must be recovered from t he experimental disease

Examples of Problems:

  • Mycobacterium leprae - requres animal host
  • Neisseria gonorrhoeae - only human host
  • Opportunistic pathogens - immunocompromised host

Use of Molecular Postulates

  • Criteria a for determining cause of infections using molecular techniques
  1. Virulence factor: gene or gene product must be found in pathogenic stains but not in non-pathogenic stains
  2. When a virulence factor is introduced into a non-pathogenic strain it should become a pathogenic strain
  3. Genes for virulence must be expressied during the course of disease
  4. Antibodies specific for the virulence gene products should be protective


Classifications of Microogranisms

Organisms:

  • Bacteria, Archaeabacteria and Eukaryotes.
  • Infectious Agents: Viruses, viroids, prions

Bacterial and fungal classification using taxonomy

  • Kingdom, Phylum, Class, Order, Family, Genus, Species
  • Order has suffix of ales, Family has suffix of aceae
  • Biovar - variant strain that differs physiologically or biochemically
  • Serovar - sub division of species
  • Strain - a genetic varient or subtype

Viral Taxonomy

  • Order suffix virales
  • Family suffix viridae
  • Subfamily suffix virinae
  • Genus suffix virus
  • Species individual virus

Bacterial Morphology

Size

Bacteria range in size between .2 micrometers and 10 micrometers , usually around 1-2 micrometeres in diameter (Mycoplasma species range from .15 -.3 micrometers in diameter Although bacteria have small size, the importance of this is their large surface area to volume ratio. Prokaryotes do not require complex nutrient uptake and transportation mechanisms (use passive diffusion)

Bacterial Shapes

  1. Spherical - Coccoid (Coccus (singular), cocci (plural)
  2. Cylindrical - Rod (Bacillus (singular), Bacilli (plural)
  3. Curved, Helical Twisted, Spiral Vibrio (refers to vibratory motility)
  4. Square Cuboid (not infectious)

Cell Arrangement

  • Bacteria can be arranged singularly, in pairs, in chains or in clusters
  • Specific Terminology is used for coccoidal Bacterial arrangements
    • Single Single
    • Pairs: Diplococci Streptococcus pneumoniae
    • Chains Streptococci Streptococcus pyogenes
    • Clusters Staphylococci All Staphlococcus species
    • Tetrads Example: Sarcina species
    • These terms do not exsist for bacillus
  • Coccobacillus - a very short bacillus, a term indicating an intermediate appearance of a bacilli and coccus
  • Pleomorphic - cannot retain proper shape or proper stain. Could be very old or very young bacteria

Staining Techniques

Grams staining

Invented in 1884 by Hans Christian gram. This is the most important staining technique for identifying and classifying bacteria.
Utilizes the ability of bacteria to retain crystal violet-iodine complex after exposure to organic solvent.
Note: Never carried out on a throat swab or fecal location.
Steps:

  1. Flood with Crystal violet (primary stain)
  2. Flood with Gram's iodine (mordant)
  3. Drop wise add decoloriser (acetone or alcohol) *critical step*
  4. Immediately rinse with water
  5. Flood with safranin (counter stain)

Gram Positive Bacteria stain Blue/Purple Gram Negative Bacteria stain Red

Utility of Gram Stains

  1. Determine the adequacy of the specimen for culture
  2. Make a presumptive etiologic diagnosis and early clinical decision
  3. Suggest a need for non-routine laboratory procedure
  4. Help make accurate interpretation of culture results
  5. Provide better insight into the current infection

Acid Fast Staining

Devised By Paul Ehrlich Used for bacteria resistant to Gram's stain ie: M. Tuberculosis and Nocardia species These two species are not gram (+) or gram (-) They are resistant to gram's stain they have a thin peptidoglycan layer with external arabinogalactan layers which are esterified into glycolipids (mycolic acids). This gives the cell wall a waxy appearance.

Ziehl-Neelsen staining (hot method)

  1. Flood with hot basic carbolfuchsin; decolorize with acid-alkali
  2. Counterstain with methylene blue or malachite green
  3. Acid fast bacteria - Red/Pink; Non Acid-fast bacteria - Blue/Green

Kinyoun Stain (cold method)

Same as Ziegl-neelsen method but does not require heat

Fluorochrome stain

(auramine-rhodamine)

  • Primary stain is a fluorescent dye and the counterstain is an oxidizing agent (Potassium permanganate)
  • The bacteria would be yellowish/green against black background

Bacterial Ultrastructure

Flagellum

Flagella (plural form)

General features
  • Extends outwards from within the cell
  • Provides motility...but some bacteria are motile but lack flagella
  • May have an essential role for colonization (contributes to virulence)
  • Not essential for bacterial survival
  • All coccoidal cells lack flagella
Flagellum arrangement

Polar (at one or both ends/poles) or Lateral(situated around the entire surface)

Composition of flagellum

Composed of 3 parts

Helical filaments
  • long & thin ~20nm dia by 1-7 micrometers in length
  • Composed of protein - flagellin, antigenic termed the H (Haugch antigen). This H-antigen is useful for identifcation of bacteria
    • <math>H^+</math> possess flagella (motile)
    • <math>H^-</math> lack flagella (non-motile)
Hook
  • a short curved structure which anchors the filament into the basal body
Basal Body
  • Contains rod and 1-2 sets of double plates/rings.
  • All flagella contain two rings (S & M) for rotation located in the cytoplasmic membrane.
  • Gram negative flagella possess additional 2 rings (L & P) located in outer membrane of cell wall which stabilize the filament)
    • 2 rings Gram positive bacteria
    • 4 rings Gram negative bacteria
Movement
  • Taxis - involuntary movement of organism in response to a stimulus
  • Magnetotaxis - magnetic field
  • Phototaxis - light
  • Thermotaxis - heat
Chemotaxis
  • response to chemical stimulus (nutrients or growth inhibitors within the environment
  • Chemicals are recognized by protein receptors located in the cytoplasmic membrane
Recognition of response
  • + response- organism swims from low concentration to high concentration (up the concentration gradient)
  • - response - organism swims down the concentration gradient
  • Non response- random walk
Mechanism
  • Rotation of basal body plates in the clockwise or counterclockwise direction
  • ccw rotation - flagella sweeps around cell in common axis which results in a forward motion
  • cw rotation - Reversal of basal plates which causes the flagella to stop rotating and results in a tumbling motion to allow for a change in direction
  • random movement indicates absence of concentration gradient
  • movement during the presence of an attractant gradient resulting in a reduced tumbling frequency.

Additional outer appendages

  • Play no role in motility
  • Hollow , helical 9-10 nm diameter, thinner, shorter and more numerous than flagella.
  • Composed of PILIN protein (antigenic, can be used for classification & identification)
Pilus/Pili
  • F-Pilus (Fertility/sex)
  • A bacterium may possess a single pilus or up to 10
  • Found only in Gram negative bacteria
  • Function: transfer of genetic material between bacterial cells
Fimbria
  • known as common attachment pili (type I pili) or Colonization factor antigens (CFA)
  • enterotoxigenic E.coli
  • Mostly gram negative bactera and some gram positive bacteria
  • Function: adhesion of bacteria to surfaces, ie: tissues helps bacteria to establish colonization at a particular site (virulence)

Glycocalyx

  • Known as the capsule or slime layer
  • External polysaccharide
  • Exception: B.anthracis has a polypeptide glycocalyx
  • non vital
  • not all bacteria possess a glycocalyx
  • shows some degree of organization
Slime layer
  • poor organization, weakly attached to cell wall and easily removed.
  • ie: Coagulase Negative Staphylococcus epidermis
  • Slime produced allows attachment to medical plastic implants leading to biofilm formation
Capsule
  • organized, tightly adhered to cell wall harder to remove
  • known as the K antigen (or M antigen in Streptococcus pyogenes or Vi antigen in Salmonella
Function
  1. Adherence in order to form colonies with other species members and to stick to surfaces
  2. Antigenic activity for id(detect K antigen)
  3. Antiphagocytosis - resists phagocytosis
  4. Prevention of neutrophil killing of engulfed bacteria by limmiting access of the lysosome contents into the interior of the bacterial cell
  5. Prevention of Polymorphonucleur (PMN) Leukocyte migration to site of infection- substance released from capsule paralyzes PMN leukocyte
  6. Toxicity to host cell
  7. Protection of bacterial cell against - oxygen toxicity (anaerobes), desiccation & nutrient loss
Quelling Reaction

a swelling reaction that detects the presence of a capsule add antiserum and capsulated bacteria -> results in swelling use specific antigens (K/M/Vi) for serotyping

Cell Wall

  • surrounds all eubacteria except mycoplasma species
  • important bacterial characteristics
  • structure and function distinctive
  • composed of peptidoglycan (murein) backbone-contains L&d amino acid isomers .This is unique to bacteria
  • composed of a diasaccharide of NAG (N-actylemuramic acid) and NAM (Nacetylglucosamine) by a Beta 1-4 glycosidic bonds
  • Provides rigidity and strength
  • prevents osmotic lysis
  • 3rd aaof one tetrapeptide to the 4th aa of a second tetra peptide on NAM on adjacent glycan backbones
  • This provides rigidity and strenthg and prevents osmotic lysis

Gram negative E coli Gram positive Ecoli 3rd aa: diaminopmelic acid 3rd aa: L-lysine direct lipprotein cross linkage Pentaglycine cross linkage few cross linkages Greater cross linkage > 90o open mesh framework A tight framework

Gram Positive Bacterial cell Wall
  • Simple composition of thick peptidoglucan layer (50-60% of weight)
  • Teichoic and lipoteichoic acids are only found in gram positive bacteria
  • these are highly anionic so they sequester cations
  • bind proteins, act as adhesive sites(adhesins) for viruses
  • are antigenic; used for id and classification
  • Cell wall may contain CH20 and proteins depending upon species
  • (M,T,R proteins of group A streptococcus, protein A of staphlyococcus aureus)
Roles of lipoteichoic acid during disease
  • dermal necrosis (schwartzman rxn)
  • induction of cell mitosis at site of infection
  • stimulate specific immunity
  • adhesion to the human cell
  • complement activation
  • induction of hypersensitivity (anaphylaxis)
Why do gram positive bacteria stain purple
  • crystal violet pierces cell wall and sits above cytoplasmic layer
  • gram's iodine passes peptidoglycan layer and sits on the cytoplasmic layer to help cell retain crystal violet
  • add decolorizer to remove the excess stain
  • peptidoglycan is hydrated
  • add organic solvent -> causess peptidoglycan to become dehydrated -> crystal violet is trapped in periplasmic space
Gram negative bacteria
  • complex composition of thin peptidoglycan layer comprimising 5-10% of bacterial cell dry weight
  • outer membrane present attached to peptidoglycan layer by lipoproteins known as braun proteins
  • membrane contains porins (protein channels) as well as lipopolysaccharide which extends outward
Lipopolysaccharide LPS structure
  • entire structure is known as endotoxin -
  • Lipid A anchors LPS to outer membrane (toxic)
  • Core polysaccharide portion external to lipid A
  • Terminal polysaccharide - repeating subunits known as O-antigen
  • provides bacterial serotypes used forclassification and identification ecoli O157:H7
=Effects of Endotoxin=
  • fever
  • hemorrhagic necrosis (schwartzman reaction )
  • disseminated intravascular coagulation
  • Production of TNF
  • Activate alternative complement pathway
  • stimulate bone marrow cell prolferation
  • enhance the immune and limulus lysate reaction
  • can lead to septic shock
Lipoligosaccharide (LOS)

Some gram negative bacteria ( Bordetella pertusis , neisseria meningitidis and camplyobacter jejuni have LPS without the O polysaccharide but with an extended core region these have a LOS Lipoligosaccharide comprises of 2 components -> no terminal repeating subunits

  1. Lipid A (toxic
  2. core oligosaccharide external to lipid A
  • virulence factor : LOS levels correlate with release of IL-1, IL-6 and TNFalpha -imp in meningococcemia and meningoccal meningitis


Gram negative bacteria stains pink/red because the thin layer of peptidoglycan, can't dehydrate much to trap crystal violet, also the crosslinkages allows gram negatives ....outer membrane layer


Chemical agents on bacterial cell walls

effects of lysozyme

produced by humans in saliva,mucus, blood sweat and tears cleaves the beta 1-4 bonds between nam and nag destroys all or part of the cell wall result -> wall degraded, w/ wall support .5 sucrose) cell will not lyse)

-spheroplast: mostly gram negative -> portion of cell wall remains
-protoplast: mostly gram positive -> cell wall completely removed

why are gram positive more susceptiel to lsozyme and penicillin than gram negative bacteria

penicilli

-binds irreversibly to penicillin binding proteins (PBPs) Transpeptidases, enzymes needed for peptidoglycan synthesis

Penicillin forms an inactive complex with the the transpeptidase inhibiting formation of peptide cross linkages to prevent complete cell wall formation. Results in defective cell walls with no protection to osmotic shock which leads to cell death -This only works on growing cells Penicillin will have no effect on mycoplasma species because they lack a cell wall


periplasmic space

space between inner and outer membranes of gram negative bacteria but is also present in some gram positive gel like area of loose network of peptidoglycans contains nutrient transport proteins, preoteases, detoxifying enzymes, membrane derived oligosaccharides and osmoprotectants

axial filaments

-flagella like filaments (similar chemically and structurally) Composed of a long thin microfibril inserted into a hook entire structure is in the periplasmic space and is referred to as the endoflagellum they have a corkscrew-like motion

cytoplasmic membrane

aka plasma membrane/inner membrane, or cell membrane

a phospholipid bilayer 4-5nm thick, phospholipid 30-40% & protein 60-70% proteins intergral and peripheral

      • no sterols except mycoplasma species

semipermeable barrier

Functions

  1. active transport
  2. secretion of extracellular enzymes and toxins
  3. oxidative phosphorylation ...bacteria do not have mitochondria (cytochromes and dehydrogenase enzymes are in the cyltoplasmic membrane
  4. biosynthesis and export cell wall components (phospholipid biosynthesis
  5. anchoring dna (during cell divison ) (mesasome)
  6. chemotactic response - receptors located wthin the cytoplasmic membrane

internal contents

  1. protoplasm/cytosol

granular appearance due to free ribosomes site of all biochemical activity of a bacterial cell composed of 70-80% water which acts as a solvent

  1. Ribosomes

RNA/protein bodies sites of protein synthesis 70S ribosomes made up of 50S and and 30S subunits eukaryotic -> 80S from 60S + 40S another site for antibiotics

  1. Mesosomes

Extensive invaginations of cytoplasmic membranes mainly seen in gram positive bacteria -unknown function, may anchor dna during cell division

  1. chromatin area

prockaryotes do not possess a distinct membrane enclosed nucleus and no mitotic apparatus DNA is aggregated in one area known as a nucleoid

  1. bacterial chromosome

-single haploid circular dna known as chromatin body -some exceptions: streptomyces and borrelia species (linear) rhodobacter sphaeroides (2 seperate chromosomes) size on average 3x10^9 daltons smaller than eukaryotes all genes are linked no histone proteins (instead have Mg+2 polyamines which acts like histones)

  1. Plasmids

circular extrachromosomal DNA smaller and seperate from chromosome capable of self replicating carry supplemental genetic information (antibiotic resistance (r-plasmid) Production of toxins Mating capabilities (F-plasmid -> F-pilus0 Tolerance to toxic metals

  1. Inclusion bodies - not always found in a bacterial cell

storage granules : energy and nutrient stores seen under light microscopy not permanent structures ie poly beta hydroybutyrate (PHN polymetaphosphate glycogen starch

  1. vegetative bacterial cells -> Bacterial cells capable of growth

Endospores

-not a structure of the bacterial cells specialized structure that enable survival produced by bacillus and clostridium species Resistant to UV, irradiation, chemical disinfection and drying require a speicalized endospore stain (Light microscopy)

structure

coat- - keratin like protein provides an impermeable layer (resistance to antibacterials) cortex - type of peptidoglycans (fewer crosslinks) spore wall peptidoglycan layer (taken from cell wall of vegetative cell) core - contains complete nucleus protein synthesizing apparatus energy-generating system (glycolysis) and calcium dipicolinic acid

differences between veg cells and endospores property vegatative cell endospore surface coat typically gram+ thick spore coat + peptidogylcan spore wall microscopic appearance non-refractile refractile Calcium dipicolic acid absent present in core Enzymatic activity present absent Heat resistance low high radiation resistance low high resistance to chemicals (acids) low high sensitivity to lysozyme sensitive resistant sensitivity to stains/dyes sensitive resistant

sporulation / sporogenesis

true differentiation ...1 veg cell -> 1 spore this is not growth takes 6-8 hours to complete sporulation

  -dna condences
- transverser wall  ??????????
- spore forms inside vegatative cell
vegatative cell grows around the spore forms multi layered container

cell lysis


germination

outgrowth from spore 1 spore -> vegatative cell only occurs when environmental conditions are suitable for bacterial cell growth takes 2 hours to complete


Atypical bacteria

Mycoplasmataceae species

Examples include:

  • Mycoplasma pneumoniae
  • Mycoplasma genitalium
  • Mycoplasma hominis
  • Ureaplasma urealyticum

Size and Shape

  • smallest known free-living organisms .15-.3 diameters
  • contain minimal set of organelles required for growth and replicaion
  • shape varies: pleomorphic (coccoid or long filaments) relates to cellular division
  • unique feature: possess no cell wall -entire life
  • do not synthesize peptidoglycan but will stain pink on gram staining
  • resistant to certain antimicrobial agents (penicillian and lysozyme)


  • cytoplasmic membrane is the external surface layer
  • typical phosphloipid ilyaer structure
  • unique feature: contains sterols

  • provide integrity and strength but allows mycoplasmataceae species suseptible to amphotericin B

Cytoplasm

only contains DNA and ribosomes, has filamantous shape


Unique feature

  • attachment organelle

in M pneumonaiea M genitalium

  • allows attachment to epithelial cells lining respiratory and urogenital tract
  • has a tapered tip coated with P1 adhesin attaches to receptors at base of cilia
  • results in tissue destruction due to hyrpogen peroxide and superoxide ion
  • absense of organelle leads to avirulence

nucleoid

  • small genome
  • smallest capable of self production encodes for a few proteins requires supplemental media . single circular strand of DNA


Rickettsiaeceae

  • includes rickettsia and orientia

size and shape

  • small .3-.5 x .8-1.0 micrometers
  • shape bacilli - coccoidal (pleomorphic)
  • closely related to Gram negatives (do not stain well) use giemsa or gimenez stain

unique features

  • obligate intracellular pathogens
  • unable to produce sufficient energy to replicate extracellulary
  • can not be cultivated on agar
  • only grow in/on cell culture embyronated eggs susceptible animals
  • b/c Possess no coenzyme A, NAD or ATP - all obtained from host
  • rickettsia - grow in cytoplasm of cells
  • zoonotic- transmitted via arthropod vectors such as ticks lice fleas mites
*  eg R prowazekii human body louse, R rickettsiae - wook tick

Anaplasmataceae and coxiella

  • family anaplasmataceae
  • medically important:
  • E chaffeensis E ewingii A phagocytophilum causing Ehrlichiosis, C.burnetti causes Q fever (cattle goats sheep)

small .2-2.0micrometers

  • obligate intracellular pathogens
  • zoonotic anaplasmataceae
  • arthropd vectors : ticks


chlamydiacea

  • chlamydia:

C trachomatis

  • chlamydophila:

C psittaci, C.pneumoniae

  • human infections only: trachoma,inclusion conjunctivitis & lymphogranuloma venereum
  • zoonotic infections (birds-> man) Parrots finches pigeons chickens ducks turkeys and seabirds

size and shape

  • small .2-.7 micrometers
  • not motile
  • coccoidal

unique feature

  • obligate intracellular pathogens
  • grow in cytoplasmic vacuoles in limited host cell types depend on host for ATP and NAD+
  • can not synthesize ATP or reoxidze NADP
  • no detectable flavoproteins or cytochromes
  • No peptidglycan detected in 1st stage of life cycle
  • complex life cycle producin g2 developmental forms
  • which is detected by gimenez and giemsa stains

Elementary Body (EB) Reticulate (RB) or Initial Body (IB) Non replicating Replicating infectious non-infectious small .25-.3 micrometers larger.5-.7 micrometers diameter adapted for extracellular survival adapted for intracellular growth induce endocytosis does not induce endocytosis metabolically inactive metabolically active


developmental cycle

  1. eb taken into host by phagocytosis
  2. next 8 hours EB reorganizes -> RB (IB)
  3. RB grows and divides by binary fision
  4. 24-48 hours RB reorganizes -> EB (forms cell wall)
  5. Compled host cell liberates EB's
    • this life cycle is unique to chlamydia*

Fungi

  • Eukaryotic: nucleus / nuclear membrane but no chorophyll
  • macroforms mushrooms puffballs gill fungi
  • microforms molds & yeasts
  • all fungi are saprobes(live on dead/decaying matter)
  • types of relationships
    • symbionts mutal advantage
    • commensals one benefits orther is neither harmed/hurt
    • parasites one benefits and is harmful to the host

Micro fungi

  • Yeasts unicellular
  • typically round/oval single cells
  • 4-5 micrometers in diameter up to 24 micrometers
  • reproduce asexually by budding
  • form elongated tubes termed pseudohypae

molds

  • multucellular
  • long filamentous/tubular structures hyphae which intertwine to form the mycelium
  • hypha range from 5-50 micrometers in length 2-4 micro in diameter
  • reproduce sexually and/or asexual by producing spores

hyphae

  • thread like branching filaments nucleated tubes of cytoplasm
  • characterised by presence/absense of wall partitions
  • septate partitioned by cross walls
  • aseptate or coenocytic not partitioned
  • differences between hyphae vs yeast pseudohyphae

feature present in permanent hyphae differentiation hyphae branching true hyphae arthrospores and chlamydospores true hyphae

Mycelium

  • intertwined mat of hyphae
  • composed of 2 portions
  • vegatative- attached to substrate penetrates to obtain substrate
  • reporductive represented by aerial structures
  • asexual reproduction propagules (conidia) (spores)

dimorphic fungi

  • yeast<->mold
  • morphological and physiological conversion of certan fungi from one phenotype to another
  • same organism can exhibit 2 different forms
  • depends on chemical and physical factors
  • important pathogenically
  • candida albicans coccidio immitis, histoplasma cap........lots


  • what are the dimorphic forms
  • freeliving state mycelial or hyphal forms
  • occurs at sub physiological temperature distinct sexual forms are displayed
  • parasitic yeast forms
  • due mainly to physiological temperature 37 oval morphology
  • most but not all fungi are dimorphic
  • aspergillus species are always in mycelial phase even in deep tissue infections
  • torula species exist only as yeast
  • candida albicans is truly dimrphic yeast human tissue
  • mycelial (mold) form in culture
  • produces pseudohypae in biofilm superficial, cutaneous and mucus membrane infections

fungal staining procedures

  • KOH preparation/procedure
  1. take scraping from margin (not center) of lesion
  2. place on clean slide
  3. add 2-4 drops of 10% KOH in water
  4. warm slide do not boil -> allows kOH to denature eukaryotic tissue
  5. optionally add 1 drop of lactophenol cotton blue
  6. add cover slip
  7. examine immediately under high dry magnification

use of staining

  • determine fungal morphology ie yeast or mould
  • if mold identify hyphae septate/nonseptate spores (thallospores or sporangium) (type of arrangement)

Fungi stain gram positive, can differentiate by size ....fungi are HUGE

fungal architecture

  • eukaryotic
    • cell surface - glycocalyx (there's a capsule in 1 species)
    • cell wall
    • cytoplasmic membrane
    • nucleus - nuclear envelope nucleolus chromosome
    • cytoplasm organelle er golgi mitochondria vaculoes
    • cytoskeleton microtubules microfilaments
    • ribosomes 80s
  • absence of locomotion structures ( no flagella)

*absense of capsules except cryptococcus neoformans************

cell wall

  • contains no peptidoglycan layer ***differs from bacteria
  • thick and rigid complex composition
  • chitin (polymer of nacetylDglucosamine) (not NAG molecule)
  • additional polysacc such as glucans and mannan
  • proteins
  • may contain lipids

cytoplasmic membrane

  • typical phospholipid bilayer but differs from bacteria
  • contains sterols ergosterol but not cholesterol
  • site of antifungal drugs
  • functions the same as all membranes

cytoplasmic contents

  • differs from bacteria
  • more complex
  • golgi body, endoplamsic reticulum cytoskeleton
  • ribosome 80S
  • microtubles spindle fibers.. tubulin - site of antifungal drug

nucleus (euk)

  • differs from bacteria
  • nuclear membrane bound (nuclear envelope)
  • most haploid
  • contains the chromosome larger than bacteria


fungal classification

  • kingdom: fungi
  • 2 divisions
  • myxomycota eumycota 5 subdivisions 4 are pathogenic to humans
  • class zygomycetes most primitive filamentous non-septate separates them from other subdivisions
  • reproduce sexually zygospores and asexually sporangiospores
  • not primary pathogens (opportunistic)
  • rhizopus mucor water molds

class ascomycetes

  • septate produce sexual ascospores and asexual conidia spores
  • members include yeasts and molds - dermatophytes (skin infections ) some aspergillus species

class basidomycetes

  • septate produce sexual spores basidospores
  • members include mushrooms and puffballs
  • only human pathogen filobasidella neoforms (sexually reproductive form of C. neoformans)

class deuteromycetes

  • septate: reproduce asexually septate hyphae sexual reprodutive structures unknown
  • includes yeasts and molds
  • ie candida coccidioides species
  • epidermophyton species
  • malassezia
  • microsporium species
  • trichophyton speicies

class Archiascomycetes

  • reproduce sexually fusion and asexually binary fission
  • only member pneumocystis jirovecii (previously known as P. Carnii)
  • Fungal disease mycoses
  • systemic by inhalation initially pulmonary---> disseminated
  • subcutaneous infections of deep layers (muscle and fascia) most by wound puncture remain localized
  • cutaneous invade keratinized and cutaneous tissue also hair shaft and nail bed
  • superficial infections of limited to surfaces of skin and hair innocuous and reappear (also nails) (rarley evoke an immune response
  • opportunistic infections debilitated and traumatized patients, immuno-suppressed
  • mycotoxins: mycetismus (amantia mushroom) afloatoxin (aspergillus flavus)

features shared with bacteria

  1. microorganisms
  2. all fungi grow axenically without others on artifical media
  3. all fungi are aerobic or facultative
  4. most fungi are not capable of invading living human tissue

fungal features different from bacteria

  1. all fungi are eukaryotic
  2. different internal organelles
  3. membrane bound nuc, chromosomes that sort by mitosis
  4. 80s ribosomes
  5. larger than bacteria
  6. biochemically different
  7. fungi grow more slowly
  8. fungi have multiple reproductive patterns usually but not always

Viruses

general characterisics

  • infectious agents
  • acellular
  • require hosts for replication
  • termed obligate intracellular parasites


  • exploit machinery of host cell
  • possess no ribosomes nor ATP generating systems
  • replicate independently of host cell chromosome

virus definition

a subcellular agent consisting of a core of nucleic acid surrounded by a protein coat that must use the metabolic machinery of a living host to replicate and produce more viral particules

viron

A term for complete infectious viral particle (extracellular form)

size

  • ultramicroscopic- smallest infectous agent <.2micrometers)
  • require electron microscope to visualize
  • animal viruses largest .4 microm in length
  • smallest .02 microm diameter

viral ultra structure

  • composition is simple by diverse varying in size shape and chemical composition
  • has a covering and a central core
  • covering -capsid and envelope(but not in all viruses)
  • central core - nucleic acid and various proteins
  • capsid found in all viruses
  • capsid protects the genome
  • protein shell surrounds nucleic acid sometimes referred to as nucleocapsid (capsid and nucleic acid)
  • composed of identifcal individual protein molecules known as protomers
  • protomers organize aggregate themselves into capsosmers polypeptides

**advantage: reduces the need for excessive genetic information and promotes self assembly requiring no ATP or additional enzymes there are two types of capsid symmetry helical and icosahedral

helical capsid

  • a viron with helical symmetry appears rod shaped . Capsomers are arranged into a coild -> helix 3d shape
  • diameter is determined by size and packaging of the protomers
  • length is determined by length of NA

iscosahedral capsid

  • a viron with icosahedral symmetry appears approximately spherical
  • shape and dimensions depend on protomers (3d)
  • in general a 20 sided polygon (12 spaced corners with a capsomer at each corner) with two capsomer types
  • triangular hexons composes the flat faces of 6 capsomers
  • round pentons compose the corners of 5 capsomers
  • there are variable capsomer #'s
  • poliovirus 32 (12 pentons , 20 hexons) adenovirus 252 (12 pentons , 240 hexons)

capsid function

  1. provides the nucleic acid protection from digestion by enzymes
  2. contains special sites on its surface allowing attachment of the virion to a host cell
  3. provides proteins that enable the virion to penetrate the host cell membrane
  4. in some to inject the infectious nucleic acid into the cell's cytoplasm

viral envelope

  • surrounds nucleocapsid of many viruses but not all
  • a protein phospholiipid layer of unique composition
  • lipids from host cell membrane
  • proteins are virus specific modified viral proteins not host membrane proteins

note sensitivity

  • presence of an envelope makes the viral particle sensitive to lipid dissolving agents (ether) dessication (influcenting survival time in the environment) detergents acids etc

envelope -> adds instability

types of envelope proteins

  • matrix proteins link envelope to capsid
  • stabilize virus and mediates interaction between capsid proteins and envelope
  • surface proteins exposed capsid proteins or envelope
  • glycoproteins sometimes reffered to as spikes or peplomers
  • requred for attachement of virus to host cell (antigenic determinants)
example of exposed surface proteins
  • influenza virus has 2 types
  • haemagglutinin (surface glycoprotein)
  • attaches to sialic acid residues of host cell surface, agglutinates RBCs
  • Neuroaminidase (surface spike) dissolves neuraminic acid (cytoplasmic membrane of mammalian cells) aids in releasing virus from host cell
  • please note not all virusses possess an envelope
  • if lacking - virus is termed a naked virus as opposed to an enveloped virus

nucleoprotein core

  • viral nucleic acid (NA) - this is either DNA or RNA but not both
  • viral genomes are small
  • largest known bacteriophage - 670kbp
  • note bacteria 1000-5000kbp
  • some genomes are so small that they only contain 4 genes (hep b virus) .. herpes contains hundreds
  • Is so small becuses viruses use host cell's nucleic acid and protein and energy so only need genes to encode for proteins of own envelope , capsid and nucleoprotein core

Viral genome types

  • there are a variety of viral genome types

dna viruses (either double ds or single stranded)

  • most we deal with are double stranded
  • for example
    • poxviridae
    • herpesviridae
    • adenoviridae
    • hepadnaviridae
    • papoviridae and polymaviruses

rna viruses

  • learn mostly single stranded ones
  • positive (+) stranded rna - genomes of the same polarity as mRNA
  • ie Picornaviridae and calciviridae
  • Negative (-) stranded RNA genomes of opposite polarity to mRNA
  • ie Rhabdoviridae
  • Unique Ambisense genomes (both positive and negative stranded RNA)
  • ie arenaviridae and bunyaviridae

nucleoproteins

  • found within the core
  • stabilize nucleic acid during replication or have enzymatic function
  • majority required for replication strategies

Polymerases (synth of dna 7 rna)

  • dna -dependent RNA polymerase (poxvirus)
  • RNA dependent DNA polymerase (RNA tumor viruses)
  • RNA dependent RNA polymerase (RNA viruses)

Reverse transcriptase

DNA copy from RNA genome (HIV)

VIRAL classification

ICTV standards

  1. type of nucleic acid
  2. symmetry of capsid
  3. presence/absense of envelope
  4. size of virus particle

baltimore system

  • production of mRNA during infection
  • compliments ICTV
  • viral replication strategies
  • placement of viruses in 7 arbitary groups
classI double stranded DNA
  • some replicate in nucleus
  • some replicate in cytoplasm
classII single stranded(+) sense DNA
  • replications occurs in the nucleus
  • formation of (-) sense strand
  • serves as template for +strand RNA and DNA synthesis
class III double stranded RNA
  • have segmented genomes
  • each genome segment transcribed separately to produce monocistronic mRNAs
classIV single stranded + sense RNA
polycistronic mRNA
  • hep A genome RNA = mRNA
  • naked RNA is infectious , no viron particle associated polymerase
  • translation-> formation of polyprotein product
  • cleaved to form mature proteins
complex transcription
  • two or more rounds of translation necessary to produce genomic RNA
Class V single stranded - sense RNA
  • must have viron particle RNA directd RNA polymerase
segmented
  • first step transcription of (-) sense rna genome by viron RNA dependant RNA polymerase to produce monocistronic mRNAs
  • serve as the template for genome replication
non-segmented
  • as above monocistronic mRNA are produced
class VI Single stranded + sense RNA with DNA intermediate in life cycle (retroviruses)
  • genome + sense but unique doploid)

vdoes not serve as mRNA but as template for reverse transcription

ClassVII double stranded DNA with RNA intermediate
  • relies on reverse transcription
  • occurs inside particle on maturation
  • on infection of new cell
  • first event is repair of gapped genome then transcription


Atypical virus like agents

  • defective- complete virion but unable to replicate without helper virus
  • pseudovirons contain host DNA not viral, infectious but no replication

Prions

  • subviral particle , no nucleic acid
  • slow infection, long incubation period (months-years)
  • transmissible spongiform encephalopathies 1950's scrapie (hseep) kuru (and now BSE bovine spongiform encephalopathy

vproion sialoglycoprotein called PrP 27-30

  • Prp gene chromosome 20
  • Prp Protein (33-35) noninfectious -> Prp (27-30)
  • link to human disease
  • neurgenerative crytzfel-jacob disease transmissible spongiform encephalopahty progressive degeneration of the brain
  • nvCJD (new variant CJD)
  • suspect for alzheimers, multiple sclerosis
  • transmission horizontal (person to person, formites, eating contaminated meat)

viroids

  • infectious agents/subviral particle
  • small circular ss RNA with/without capsid or envelope
  • mainly cause plant diseases recently identified hep D as a viroid
  • hep d is enclose in a hep b virus capsid
    • must be simulataneously infected with both hep b and hep D viroid

bacterial viruses

  • bacteriophage/phage is a viral agent of bacteria

structure

  • phage head : protein capsid (icosahedral symmetry) which surrounds nucleic acid (majority ds DNA)
  • tail: attached prtein (helical in structure which may have base end plate with spikes (pins and tail fibers which allows injection of nucleic acid in bacterial cell

Comaprision

Character Bacteria Mycoplasma Chlamydiae Rickettsiae Fungi Viruses DNA Both Both Both Both Both Both Obligate intracellular pathogen No No Yes Yes Maybe Yes Peptidoglycan cellwall Yes No Yes (1 stage) Yes No No Growth on artifical media Yes Ys No No Yes No Contain ribosomes Yes Yes Yes Yes Yes No

Micobial gowth and multiplication

  • Physical and chemical requirements are required to grow and multiply
  • Physical requirements such as temperature osmotic pressure and pH
  • Chemical requirements Carbon Nitrogen Sulfur phosphorus trace elements oxygen organic growth factors
  • Survival of bacteria in a hostile environment requires that there must be a continous macromolecular synthesis and genome replication. -> increase in cell mass and in bacterial genome necessitiates that the cell mst divide to maintain its physical size and the size of its genome
  • Therefore one bacterial cell divides into 2 daughter cells by asexual process known as binary fission
  • The time required for a single cell to divide into 2 daughter cells is termed as doubling time
  • doubling time is deined as formation of new daughter bacterial cells from parent cell. its growth to propersize due to replication fo bacterial macromolecules and eventual division into two cells

Sample doubling times :

  • Bacillus stearothermophilus 11min
  • Escherichia coli 20 min
  • Staphylococcus aureus 28min
  • Lactovacillus acidophilus 60-80 min
  • mycobacterium tuberculosis 360 minutes
  • treponema pallidum (causative agent of syphyllis) 1980 minutes

growth curve

in a closed system where nutrients and space are finite no removal of waste products

lag phase

newly inoculated cells need adjustment to the new environment (nutrients temp ph) 2) no cell divisiion 3) very few bacterial cells population is sparse/dilute

exponential (log) phase

1) cell adjustment is complete metabolism and replication at full speed growth at geometric / logarithmic rate cells reach maximum rate of cell division (this continues as long as nutrients and environment is favorable)

stationary phase

1) population reaches maximum numbers ate of cell inhibition (death) = rate of multiplication factors: nutrient depletion, ph changes, accumulation of waste, reduced O2 (can induce endospores of bacillus and clostridium species)

death

1) decline in growth rate 2) death in geometric proportions causes : depletion of nutrients, O2, excretion of toxic waste products, increased density of cells (limited space) factors same as stationary phase+ release of lytic enzymes

measurement of viable bacteria

serial dilution: dilute concentrated samble 10 folds and meansure enumerated bacteria in each plate count

plate count use for enumeration of viable bacteria (CFU colony forming unit) . A fraction of each dilution is placed on agar plate and counted. Assume each colony is formed by one bacterium and total # of viable bacteria = total number of colonies. Total viable count= # of colonies x sample size (.1) and the dilution factor.

Filtration; for water testing and highly diluted samples Turbitidy for blood cultures

Physical factors affecting growth

-temperature- whether a bacteria multiplies slowly or luxuriantly at certain temperatures 3 groups minimum - lowest temp at which bacteria can survive and permit microbial growht and metabolism maximum - highest temp at which bacteria can survive and permit microbial growht and metabolism optimum - narrow range of temp that promotes the fastest growth of a bacterial species

 	min 	max 	optimum

Ecoli 15-20 45 37


psychrophile

psychrophilic opt temp~ 4 celcius capable of growth at 0 degrees celcius not involved in human infections found in snow fields polar ice deep ocean examples: psydomonas, flavobacterium, alcaligenes, achromobacte species

Facultative psychrophile

grow slowly in cold condition but optimial above 20c staph aureus c. monocytogenes contamination of food/dairy


mesophile

opt temp 20-40c capable of growth 10-50 group containing human pathogens (30-37) example: e. coli

thermophile

optimum temp 60 cel. capable of growth 45-85 celcius incapable of growth at usual body temp not involved in human infections found volcano, direct exposure to sun

osmotic pressure (water activity)

all bacteria require water for growth and reproductions . It is an essential solvent involved in all biochemical reactions and indirectly maintain osmotic pressure increase of salts leads to a decrease in the rate of growth , cell swells , replication halts aw = index of free available water= atmospheric measure , relative humidity = rh rh= 1.00 aw therefore 90% rh = .9 Aw

absorption and solution factors reduce availabilty of free water (decreased Aw) pure distilled water (Aw =1) Saturated solution of NaCl (Aw=.8) Seawater NaCla = 3% most bacteria

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