Transcript Classification of Microorganisms

Lesson 4: Classification of
Microorganisms
February 3, 2015
Taxonomy
Taxonomy—the science of classifying organisms
(taxa—categories of organisms)
• Provides a reference for identifying organisms
• Carlos Linnaeus introduced a formal system of
classification, dividing living organisms into two
groups, Plantae and Animalia
– Used Latin names to provide a “common” language
for all organisms
Three Domain Classification
• Study of ribosomes allowed scientists to divide cell
types into three groups
– Remember ALL cells contain ribosomes
– rRNA (16S RNA) are different for each group
• Eukarya, Bacteria, and Archaea
– Bacteria and Archaea are very similar
• Bacteria contains peptidoglycan and Archaea do not
• Molecular Clock measures evolution in terms of
mutations in the nucleotide sequences
– rRNA sequences mutate slowly (highly conserved) and
provide the most confident results when looking at
evolution
Clinical Application
• Several classes of antibiotics work by
inhibiting protein synthesis
– The disruption of protein synthesis leads to the
death of a cell
• Streptomycin and Gentamicin attaches to the
30S subunit and Erythromicin and
Chloramphenicol attaches to the 50S subunit
• Antibiotics do not interfere with Eukaryotic
protein synthesis
Figure 10.1 The Three-Domain System.
Eukarya
Fungi
Origin of mitochondria
Bacteria
Animals
Amebae
Origin of chloroplasts
Mitochondria
Slime molds
Archaea
Cyanobacteria
Proteobacteria
Chloroplasts
Methanogens
Plants
Extreme
halophiles
Ciliates
Green
algae
Dinoflagellates
Diatoms
Hyperthermophiles
Gram-positive
bacteria
Euglenozoa
Thermotoga
Giardia
Horizontal gene transfer
occurred within the
community of early cells.
Mitochondrion degenerates
Nucleoplasm grows larger
Lateral Gene Transfer
Vertical Gene Transfer
Table 10.1 Some Characteristics of Archaea, Bacteria, and Eukarya
Phylogenetics
• Each species retains some characteristics of
its ancestor
• Grouping organisms according to common
properties implies that a group of organisms
evolved from a common ancestor
1. Anatomy (Gram stain, Flagella, Shape)
2. Metabolic Processes (Oxygen usage, Fermenter)
3. rRNA
Scientific Nomenclature
• Common names
– Vary with languages and with geography
– Spanish Moss
• Tillandsia usneiodes
• Binomial nomenclature (genus + specific
epithet)
– Used worldwide
– Genus capitalized and species lowercase
• Escherichia coli and Homo sapiens
Scientific Names
Scientific Binomial
Source of
Genus Name
Source of
Specific Epithet
Klebsiella pneumoniae
Honors Edwin Klebs
The disease
Pfiesteria piscicida
Honors Lois Pfiester
Disease in fish
Salmonella typhimurium Honors Daniel Salmon Stupor (typh-) in
mice (muri-)
Streptococcus
Chains of cells
Forms pus (pyo-)
pyogenes
(strepto-)
Penicillium
chrysogenum
Tuftlike (penicill-)
Produces a yellow
(chryso-) pigment
Trypanosoma cruzi
Corkscrew-like
(trypano = borer;
soma = body)
Honors Oswaldo
Cruz
Taxonomic Hierarchy
Domain
Kingdom
Phylum
Class
Order
Family
Genus
Species
Figure 10.5 The taxonomic hierarchy.
All organisms
Eukarya
Archaea
Bacteria
Domain
Fungi
Kingdom
None assigned for archaea
Ascomycota
Euryarcheota
Proteobacteria
Hemiascomycetes
Methanococci
Gammaproteobacteria
Saccharomycetales
Methanococcales
Enterobacteriales
Saccharomycetaceae
Methanococcaceae
Enterobacteriaceae
Phylum
Class
Order
Family
Genus
Species
None assigned for bacteria
Saccharomyces
S. cerevisiae
Baker’s yeast
Methanothermococcus
M. okinawensis
Methanococcus
Escherichia
E. coli
E. coli
Critical Thinking
• When writing the genus and species for the
first time in a paper the FULL genus must be
used. Why?
• Escherichia coli and Entamoeba coli
Classification of Prokaryotes
• Prokaryotic species: a population of cells with
similar characteristics
– Culture: grown in laboratory media
– Clone: population of cells derived from a single cell
• All cells in a clone SHOULD be identical but mutations may
occur
– Strain: genetically different cells within a clone
• Usually denoted by numbers, letters, or names that follow
the specific epithet
• E. coli O104:H4 (PATHOGENIC) VS. E. coli K12 (nonpathogenic)
Classification of Eukaryotes
• Eukaryotic species: a group of closely related
organisms that breed among themselves
• Categorized based on unicellular (Protista and
some Fungi) or multicellular (Animalia, Plantae,
and some Fungi)
• Protists may be classified into clades which are
genetically related groups (similar to strains of
bacteria)
Classification of Eukaryotes
• Animalia: multi-cellular; no cell walls;
chemoheterotrophic
• Plantae: multi-cellular; cellulose cell walls;
usually photoautotrophic
• Fungi: chemoheterotrophic; unicellular or
multicellular; cell walls of chitin; develop from
spores or hyphal fragments
• Protista: a catchall kingdom for eukaryotic
organisms that do not fit other kingdoms
– Grouped into clades based on rRNA
Classification of Viruses
• VIRUSES ARE NOT ALIVE!
– Are not comprised of cells
– Require a host to live and reproduce (obligate
intracellular parasites)
• Viral species: population of viruses with
similar characteristics that occupies a
particular ecological niche
Classification and Identification
Microorganisms
• Classification: placing organisms in groups of
related species
– Lists of characteristics of known organisms
• Identification: matching characteristics of an
“unknown” organism to lists of known organisms
– Clinical lab identification
– Microorganisms are identified for practical purposes
such as determining treatment for infection
Clinical Identification Methods
• Morphological characteristics: useful for
identifying eukaryotes but can be used for
prokaryotes
– Shapes of bacterium; colony characteristics
• Differential staining: Simple staining, Gram
staining, and acid-fast staining
– Based on cell membrane differences
• Biochemical tests: determines presence of
bacterial enzymes
– Catalases, peroxidases, agglutination tests,
fermentation tests, etc.
Figure 10.8 The use of metabolic characteristics to identify selected genera of enteric bacteria.
Can they
ferment lactose?
No
Yes
Can they use
citric acid as their
sole carbon source?
Can they use
citric acid as their
sole carbon source?
No
Shigella:
produces lysine
decarboxylase
Yes
No
Yes
Can they
ferment
sucrose?
Salmonella:
generally
produces H2S
No
Escherichia spp.
Do they
produce
acetoin?
Yes
E. coli O157
No
Citrobacter
Yes
Enterobacter
Figure 10.9 One type of rapid identification method for bacteria: Enterotube II from Becton Dickinson.
One tube containing media for 15 biochemical tests
is inoculated with an unknown enteric bacterium.
Citrate
Urease
2
Dulcitol
Phenylalanine
Sorbitol
4 +
V–P
Arabinose
Lactose
Adonitol
H2S
Indole
Ornithine
Lysine
Glucose
Gas
After incubation, the tube is observed for results.
The value for each positive test is circled, and the
numbers from each group of tests are added to give
the ID value.
2+1 4 + 2 + 1
2
Comparing the resultant ID value with a
computerized listing shows that the organism in the
tube is Proteus mirabilis.
4 + 2 + 1
1
0
+
1
4 + 2 + 1
0
7
ID Value
Organism
Atypical Test
Results
Confirmatory
Test
21006
Proteus mirabilis
Ornithine–
Sucrose
21007
Proteus mirabilis
Ornithine–
21020
Salmonella choleraesuis
Lysine–
Book References
Bergey’s Manual of Determinative
Bacteriology
Provides identification schemes for
identifying bacteria and archaea
Morphology, differential
staining, biochemical tests
Bergey’s Manual of Systematic
Based on rRNA sequencing
Bacteriology
Provides phylogenetic information on
bacteria and archaea
Serology
• Serology is the science that studies serum and
immune responses that are evident in serum
(does not contain blood cell or clotting factors)
• Combine known anti-serum plus unknown
bacterium
– Rabbit immune system injected with pathogen
produces antibodies against that pathogen
• Strains of bacteria with different antigens are
called serotypes, serovars, biovars
• Slide agglutination test
Figure 10.10 A slide agglutination test.
Positive test
Negative test
ELISA
• Enzyme-linked immunosorbent assay
– Direct or Indirect
• Direct ELISA looks for the presence of
bacterium in the serum
• Indirect ELISA looks for the presence of
antibodies in the serum
• Both use antibodies linked to enzyme
– Enzyme contains substrate that produces color
Figure 18.14.4 The ELISA method.
4
Enzyme's substrate ( ) is
added, and reaction produces
a product that causes a
visible color change ( ).
(a) A positive direct ELISA to detect
antigens
4
Enzyme's substrate ( ) is
added, and reaction produces
a product that causes a
visible color change ( ).
(b) A positive indirect ELISA to detect
antibodies
Figure 10.12 The Western blot.
If Lyme disease is suspected in a patient: Electrophoresis is used
to separate Borrelia burgdorferi proteins in the
serum. Proteins move at different rates based on their charge and
size when the gel is exposed to an electric current.
Lysed
bacteria
Polyacrylamide
gel
Proteins
Larger
Paper towels
The bands are transferred to a nitrocellulose filter by
blotting. Each band consists of many molecules of a
particular protein (antigen). The bands are not visible at
this point.
Smaller
Sponge
Salt solution
Gel
Nitrocellulose
filter
The proteins (antigens) are positioned on the filter
exactly as they were on the gel. The filter is then
washed with patient’s serum followed by anti-human
antibodies tagged with an enzyme. The patient
antibodies that combine with their specific antigen are
visible (shown here in red) when the enzyme’s
substrate is added.
The test is read. If the tagged antibodies stick to the
filter, evidence of the presence of the microorganism in
question—in this case, B. burgdorferi—has been found
in the patient’s serum.
Figure 10.13 Phage typing of a strain of Salmonella enterica.
Bacteriophages are viruses that infect bacteria
Flow Cytometry
• Uses differences in electrical conductivity
between species
• Fluorescence of some species
• Cells selectively stained with antibody plus
fluorescent dye
Figure 18.12 The fluorescence-activated cell sorter (FACS).
Fluorescently
labeled cells
1
A mixture of cells is
treated to label cells
that have certain
antigens with
fluorescent-antibody
markers.
2
Cell mixture leaves
nozzle in droplets.
3
Laser beam strikes
each droplet.
Laser beam
Detector of
scattered light
Laser
Electrode
4
Fluorescence detector
identifies fluorescent
cells by fluorescent
light emitted by cell.
5
Electrode gives
positive charge to
identified cells.
6
As cells drop between
electrically charged
plates, the cells with
a positive charge
move closer to the
negative plate.
7
The separated cells
fall into different
collection tubes.
Fluorescence
detector
Electrically
charged
metal plates
Collection
tubes
6
Genetic Identification
• DNA fingerprinting
– Electrophoresis of restriction enzyme digests
• rRNA sequencing
• Polymerase chain reaction (PCR)
Figure 10.14 DNA fingerprints.
1
2
3
4
5
6
7
Figure 10.15 DNA-DNA hybridization.
Organism A DNA
Organism B DNA
1
Heat to separate strands.
2
3
4
Combine single
strands of DNA.
Cool to allow renaturation
of double-stranded DNA.
Determine degree
of hybridization.
Complete hybridization:
organisms identical
Partial hybridization:
organisms related
No hybridization:
organisms unrelated
Figure 10.16 A DNA probe used to identify bacteria.
Plasmid
Salmonella
DNA
fragment
1
A Salmonella DNA
fragment is cloned in
E. coli.
3 Unknown bacteria
are collected
on a filter.
4
2
Cloned DNA fragments are marked with
fluorescent dye and separated into single
strands, forming DNA probes.
6
7
DNA probes are added
to the DNA from the
unknown bacteria.
DNA probes hybridize with
Salmonella DNA from sample. Then
excess probe is washed off.
Fluorescence indicates presence of
Salmonella.
The cells are lysed,
and the DNA
is released.
5
The DNA is separated into
single strands.
Fluorescent probe
Salmonella DNA
DNA from
other bacteria
Figure 10.17ab DNA chip.
(a) A DNA chip can be manufactured to
contain hundreds of thousands of synthetic
single-stranded DNA sequences. Assume
that each DNA sequence was unique to a
different gene.
(b) Unknown DNA from a sample is separated into single strands, enzymatically cut,
and labeled with a fluorescent dye.
Figure 10.17cd DNA chip.
(c) The unknown DNA is inserted into the chip and
allowed to hybridize with the DNA on the chip.
(d) The tagged DNA will bind only to the
complementary DNA on the chip. The bound DNA
will be detected by its fluorescent dye and analyzed
by a computer. In this Salmonella antimicrobial
resistance gene microarray, S. typhimurium-specific
antibiotic resistance gene probes are green, S.
typhi-specific resistance gene probes are red, and
antibiotic-resistance genes found in both serovars
appear yellow/orange.
FISH
• Fluorescent in situ hybridization
– Used to identify specific sequences in
DNA/Chromosomes
• Add DNA probe for S. aureus
Figure 10.18 FISH, or fluorescent in situ hybridization.