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.

BacteriaMitochondria

Cyanobacteria

Chloroplasts

Thermotoga

Gram-positivebacteria

Proteobacteria

Horizontal gene transferoccurred within thecommunity of early cells.

Nucleoplasm grows larger

Mitochondrion degenerates

Giardia

Euglenozoa

Diatoms

Dinoflagellates

Ciliates

AnimalsFungi

Amebae

Slime molds

Plants

Greenalgae

Eukarya

Extremehalophiles

Methanogens

Hyperthermophiles

Origin of chloroplasts

Origin of mitochondria

Archaea

Vertical Gene TransferLateral 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 ancestor1. 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 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 pyogenes

Chains of cells (strepto-)

Forms pus (pyo-)

Penicillium chrysogenum

Tuftlike (penicill-) Produces a yellow (chryso-) pigment

Trypanosoma cruzi Corkscrew-like (trypano = borer; soma = body)

Honors Oswaldo Cruz

Scientific Names

Taxonomic HierarchyDomain

KingdomPhylum

ClassOrder

FamilyGenus

Species

Fungi

Figure 10.5 The taxonomic hierarchy.

Eukarya

Ascomycota

Hemiascomycetes

Saccharomycetales

Saccharomycetaceae

Saccharomyces

S. cerevisiae

Baker’s yeast

Species

Genus

Family

Order

Class

Phylum

Kingdom

Domain

All organisms

M. okinawensis

Methanothermococcus

Methanococcaceae

Methanococcales

Methanococci

Euryarcheota

None assigned for archaea

Archaea

Methanococcus E. coli

E. coli

Escherichia

Enterobacteriaceae

Enterobacteriales

Gammaproteobacteria

Proteobacteria

None assigned for bacteria

Bacteria

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 (non-

pathogenic)

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 IdentificationMicroorganisms

• 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 theyferment lactose?

Can they usecitric acid as their

sole carbon source?

Can they usecitric acid as their

sole carbon source?

Can theyfermentsucrose?

Do theyproduceacetoin?

Escherichia spp. E. coli O157 Citrobacter Enterobacter

Shigella:produces lysinedecarboxylase

Salmonella:generally

produces H2S

No Yes

No YesNo Yes

No Yes No Yes

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.

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.

Glu

cose

Gas

Lys

ine

Orn

ithin

e

H2S

Indo

le

Ado

nito

l

Lac

tose

Arab

inos

e

Sor

bito

l

V–P

Dul

cito

l P

heny

lala

nine

Ure

ase

Citr

ate

2 + 1 4 + 2 + 1 4 + 2 + 1 4 + 2 + 1 4 + 2 + 1

1 2 0 0 7

ID Value

21006

21007

21020 Salmonella choleraesuis

Organism Atypical TestResults

ConfirmatoryTest

Proteus mirabilisProteus mirabilis Ornithine–

Ornithine–

Lysine–

Sucrose

Comparing the resultant ID value with a computerized listing shows that the organism in the tube is Proteus mirabilis.

Bergey’s Manual of Determinative BacteriologyProvides identification schemes for identifying bacteria and archaea

Morphology, differential staining, biochemical tests

Bergey’s Manual of Systematic BacteriologyProvides phylogenetic information on bacteria and archaea

Based on rRNA sequencing

Book References

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

Positive test

Figure 10.10 A slide agglutination 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.

Enzyme's substrate ( ) is added, and reaction produces a product that causes a visible color change ( ).

Enzyme's substrate ( ) is added, and reaction produces a product that causes a visible color change ( ).

(a) A positive direct ELISA to detectantigens

4 4

(b) A positive indirect ELISA to detectantibodies

Figure 10.12 The Western blot.

Lysedbacteria

Polyacrylamidegel

Proteins

Sponge

Paper towels

Salt solution

Gel

Nitrocellulosefilter

Smaller

Larger

If Lyme disease is suspected in a patient: Electrophoresis is used to separate Borrelia burgdorferi proteins in theserum. Proteins move at different rates based on their charge and size when the gel is exposed to an electric current.

The bands are transferred to a nitrocellulose filter byblotting. Each band consists of many molecules of aparticular protein (antigen). The bands are not visible atthis point.

The proteins (antigens) are positioned on the filterexactly as they were on the gel. The filter is thenwashed with patient’s serum followed by anti-humanantibodies tagged with an enzyme. The patientantibodies that combine with their specific antigen arevisible (shown here in red) when the enzyme’ssubstrate is added.

The test is read. If the tagged antibodies stick to thefilter, evidence of the presence of the microorganism inquestion—in this case, B. burgdorferi—has been foundin 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).

Collectiontubes

Laser beam strikeseach droplet.

Fluorescencedetector

LaserLaser beam

Fluorescentlylabeled cells

The separated cellsfall into differentcollection tubes.

As cells drop betweenelectrically chargedplates, the cells witha positive chargemove closer to thenegative plate.

Electrode givespositive charge toidentified cells.

Fluorescence detectoridentifies fluorescentcells by fluorescentlight emitted by cell.

Electricallychargedmetal plates

Cell mixture leavesnozzle in droplets.

A mixture of cells istreated to label cellsthat have certainantigens withfluorescent-antibodymarkers.

Detector ofscattered light

1

2

3

4

5

6

6

7

Electrode

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

Organism A DNA

Figure 10.15 DNA-DNA hybridization.

Heat to separate strands.

Organism B DNA

Determine degreeof hybridization.

Cool to allow renaturationof double-stranded DNA.

Combine singlestrands of DNA.

Complete hybridization:organisms identical

Partial hybridization:organisms related

No hybridization:organisms unrelated

1

2

3

4

Plasmid

Figure 10.16 A DNA probe used to identify bacteria.

SalmonellaDNAfragment

A Salmonella DNAfragment is cloned in E. coli.

Cloned DNA fragments are marked with fluorescent dye and separated into single strands, forming DNA probes.

Unknown bacteriaare collectedon a filter.

The cells are lysed,and the DNAis released.

The DNA is separated intosingle strands.

DNA probes are addedto the DNA from theunknown bacteria.

Fluorescent probe

Salmonella DNA

DNA fromother bacteria

DNA probes hybridize with Salmonella DNA from sample. Then excess probe is washed off. Fluorescence indicates presence of Salmonella.

1

2

6

7

3

4

5

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.