Transcript MLAB 2434 * CLINICAL MICROBIOLOGY SUMMER, 2005 CECILE

MLAB 2434 –
CLINICAL MICROBIOLOGY
KERI BROPHY-MARTINEZ
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics
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Taxonomy
 Defined as the orderly classification & grouping of
organisms into categories
 Based on genotype and phenotype
 Kingdom, Division, Class, Order, Family, Tribe,
Genus and Species ( these are the formal levels of
classification)
• Family = “Clan”; has “–aceae” ending
• Genus = “Human last name”
• Species = “Human first name”
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)

Nomenclature
 Family names: Capitalized with “aceae” endings
 Genus names: Capitalized
• When in print, genus and species are italicized.
(Staphylococcus aureus)
• When written, genus and species are underlined.
(Staphylococcus aureus)
 Species names
• Staphylococcus sp. is used when referring to the genus as a
whole when the species is not identified.
• Can be referred to as singular or plural
• “sp.” – singular (Staphylococcus sp.)
• “spp.” – plural (Staphylococcus spp.)
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Abbreviations
• First letter of the genus, followed by a period and the
species epithet
• Ex: Staphylococcus aureus changes to S. aureus
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
Bacteria Identification – test each
bacterial culture for a variety of
metabolic characteristics and
compare the results with known
results.
 All organisms are either
“prokaryotes”, “eukaryotes”, or
“archaeobacteria”
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Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)

PROKARYOTES bacteria
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Do not have a
membrane-bound
nucleus
DNA is a single
circular chromosome
Have both cell
(plasma) membrane
AND cell wall.
F= flagellum
C=capsule
P= pili
N= nuclear info
R=ribosome
CM= cytoplasmic membrane
CW= cytoplasmic wall
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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EUKARYOTES fungi, algae,
protozoa, animal
cells, and plant cells
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Cells have nuclei
that contains DNA
and are complex
Most cells do NOT
have a cell wall
V=Vesicle
M=Mitochondria
G= Golgi
NM= nuclear membrane
N= nucleus
NC= nucleolus
RER= rough endoplasmic reticulum
PM= plasma membrane
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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Archaeobacteria
 Resembles
eukaryotes
 Found in
microorganisms that
grow under extreme
environmental
conditions
 Cell wall lacks
peptidoglycan
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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Bacterial Cell Wall
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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Gram Positive (GP) Cell Wall
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Very thick protective peptidoglycan layer
Many GP antibiotics act by preventing synthesis of
peptidoglycan
Consists of cross-linked chains of glycan
Also contain teichoic acid and lipoteichoic acid
these unique structures makes these bacteria GP
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)

Gram Negative (GN) Cell Wall
Two layers; outer is much thinner
than GP cell walls
 Outer wall contains several
molecules, including Lipid A which is
responsible for producing fever and
shock in infections with GN bacteria

The Cell Wall
Gram Positive
Gram Negative
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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GP cocci in clusters
→
GN bacilli (rods) →
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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Acid Fast Cell Wall – mainly
Mycobacteria and Nocardia
Have a GP cell wall structure but
also a waxy layer of glycolipids and
fatty acids (mycolic acid)
 Waxy layer makes them difficult to
gram stain
 Cannot be decolorized by acidalcohol, hence the name “acid fast”
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Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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Absence of Cell Wall – mainly Mycoplasma
and Ureaplasma
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Lack of cell wall results in a variety of
shapes microscopically
Contain sterols in cell membrane
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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Some bacteria
produce a capsule
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Protect the
bacteria from
phagocytosis
Capsule usually
does not stain,
but can appear
as a clear area
(halo-like)
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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Some bacteria produce slime layers
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Made of polysaccharides
Inhibit phagocytosis
Aid in adherence to host tissue or
implants
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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Cell Appendages
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Flagella – exterior protein filaments that
rotate and cause bacteria to be motile
• Polar
• Extend from one end
• Can occur singly or in multiple tufts
• Peritrichous
• Flagella found on all sides of bacteria
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Pili (fimbriae) – hairlike projections that
aid in attachment to surfaces
Examples of Flagella
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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Microscopic Shapes
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Cocci (spherical)
Bacilli (rod-shaped)
Spirochetes (helical)
Groupings
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Singly
Pairs
Clusters
Chains
Palisading
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)

Size and length
Short
 Long
 Filamentous
 Fusiform
 Curved
 Pleomorphic
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Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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Common Bacterial Stains
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Gram Stain:
• cell wall structure determines
the staining characteristics
• Procedure to be covered in
lab
Acid-fast
• stains bacteria with high lipid
and wax content in their cell
walls
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)

Acridine Orange
• stains nucleic acid
of both G+ and Gbacteria, either
living or dead
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used to locate
bacteria in blood
cultures and other
specimens where
background
material obscures
gram stains
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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Methylene Blue
 stain for
Corynebacterium
diphtheriae to show
metachromatic
granules and as
counter-stain in
acid-fast stain
procedures
Lactophenol Cotton Blue
 fungal stain
Calcofluor White
 fungal stain
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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India Ink – negative stain for
capsules, surrounds certain yeasts
I NEED A BREAK!!!!!
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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Microbial Growth and Nutrition
Needs
Source of carbon for making cellular
constituents
 Source of nitrogen for making
proteins
 Source of energy (ATP) for cellular
functions
 Smaller amounts of other molecules
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Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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Nutritional Requirements for
Growth
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Autotrophs (lithotrophs)
• Able to grow simply, using only CO2,
water and inorganic salts
• Obtain energy via photosynthesis or
oxidation of inorganic compounds
• Occur in nature and do not normally
cause disease
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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Heterotrophic
• Require more complex substances for
growth
• Require an organic source of carbon and
obtain energy by oxidizing or
fermenting organic substances
• All human bacteria fall in this category
• Within this group, nutritional needs
vary greatly
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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Types of Growth/Culture Media
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Minimal medium – simple; not usually used
in diagnostic clinical microbiology
Nutrient medium – made of extracts of
meat or soy beans
Enriched medium – nutrient medium with
extra growth factors, such as blood which
encourages small numbers of organisms to
flourish
Broths- used to detect small numbers of
aerobes, anaerobes and microaerophiles
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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Nonselective medium- supports growth of
most nonfastidious microbes.
Selective medium – contains additives
that inhibit the growth of some bacteria
while allowing others to grow
Differential medium – contains additives
that allow visualization of metabolic
differences in bacteria
Transport medium – holding medium to
preserve those bacteria present but does
not allow multiplication
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
Selection of primary culture media
will vary from lab to lab
 Selection of primary media will
depend on anatomical site
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Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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Environmental Factors Influencing Growth
 pH – most media is between 7.0 and 7.5
 Temperature – most pathogens grow at
body temperature; grown at 35° C in the
lab
• Psychrophiles: cold temperatures
• 10-20o C
• Mesophiles moderate temperatures
• 20-40 o C
• Thermophiles: high temperatures
• 50-60 o C
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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Gaseous composition
• Obligate aerobes – require
oxygen
• Obligate anaerobes – cannot
grow in the presence of oxygen
• Facultative anaerobes – can
grow with or without oxygen
• Microaerophilic- grow better in
low oxygen environments ( about
20%)
• Aerotolerant anaerobes- grows
better in the absence of oxygen
• Capnophilic – grow better with
extra CO2 (5-10%)
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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Bacterial Growth
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Reproduce by
binary fission
Can be fast (as
little as 20
minutes for E.
coli or slow as
24 hours for M.
tuberculosis
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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Determination of Numbers
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Direct counting under microscope
• Estimates number of live and dead cells
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Direct plate count
• Determines the number of CFU (colony-forming
units) in broth cultures and urine cultures
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Density measurement
• Useful to prepare inoculums for antimicrobial
susceptibility testing
Is It Time For a Break?
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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Bacterial Biochemistry and Metabolism
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Metabolic reactions cause production of
energy in form of ATP
Identification systems analyze unknown
specimens for:
• Utilization of variety of substances as a source
of carbon
• Production of specific end products from
various substrates
• Production of acid or alkaline pH in the test
medium
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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Fermentation and Respiration
(Oxidation)
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Fermentation
• Anaerobic process in obligate and
facultative anaerobes
• The electron acceptor is an organic
compound
• Does NOT require oxygen
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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Respiration (Oxidation)
• More efficient energy-generating
process
• Molecular oxygen is the final electron
acceptor
• Aerobic process in obligate aerobes and
facultative anaerobes
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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Metabolic Pathways
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Embden-Meyerhoff-Parnas
• Primary cycle for bacteria
• Convert glucose to pyruvic acid, a key
intermediate
• Generates energy in the form of ATP
Pentose Phosphate pathway
 Entner-Doudoroff pathway
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Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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From pyruvic acid:
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Alcoholic fermentation
(ethanol)
Homolactic fermentation
(lactic acid)
Heterolactic fermentation
(lactic acid, CO2, alcohols,
formic and acetic acids
Propionic acid (propionic acid)
Mixed acid fermentation
(lactic, acetic, succinic, and
formic)
Butanediol
fermentation(acetoin and 2,3
butanediol)
Butyric acid fermentation
(butyric)
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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Main oxidative pathway is the Krebs
Cycle, resulting in acid and CO2
Carbohydrate Utilization & Lactose
Fermentation
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“Sugars” = carbohydrates
Lactose fermentation – key component in
identification schemes
Lactose is converted to glucose, so ALL
lactose fermenters also ferment glucose
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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Genetic Elements and Alterations
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Plasmid
• Extra piece of DNA
• Code for antibiotic resistance and
other virulence factors are often found
on plasmids
• Sometimes passed from one bacterial
species to another. This is how
resistance is acquired.
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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Plasmid
replication
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Three methods
• Transformation
• Transduction
• Conjugation
Bacterial Cell Structure, Physiology,
Metabolism, & Genetics (cont’d)
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Mutations
“They don’t always read the book”
 Changes that occur in the DNA code
 Results in changes in the coded
protein or in the prevention of its
synthesis
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References
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Engelkirk, P., & Duben-Engelkirk, J. (2008). Laboratory Diagnosis of
Infectious Diseases: Essentials of Diagnostic Microbiology . Baltimore, MD:
Lippincott Williams and Wilkins.
http://andyannie.pbworks.com/w/page/5454436/Reproduction
http://animals.howstuffworks.com/fish/eels-slippery1.htm
http://fanaticstars.wordpress.com/2009/04/01/on-break/
http://www.istockphoto.com/stock-illustration-82213-coffee-break.php
http://pathmicro.med.sc.edu/infectious%20disease/infectious%20disease%2
0introduction.htm
http://realneo.us/content/yellowstone-national-park-hit-swarm-earthquakes
Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of Diagnostic
Microbiology (4th ed.). Maryland Heights, MO: Saunders.