Taxonomy2009 1

Taxonomy,
Classification &
Identification
Narelle George
Microbiology, Queensland Health Pathology
Service, QHPS-Central
AIMS
◆ Be able to define and explain differences between

▼ taxonomy, classification, identification
◆ Define the concept of numerical taxonomy
◆ List features used in classification systems
◆ Understand the rules for nomenclature
◆ Understand differences between identification systems
S. T. Cowan 1971
“Taxonomy is written by taxonomists in this form the

subject is so dull that few non-taxonomists are
tempted to read it and even fewer are tempted to try
it!
It is the most subjective branch of biological science

and is more an art than a science”
DEFINITIONS
◆ Taxonomy
- study of the biological relationships between
organisms
◆ Classification
- the orderly arrangement of units
◆ Nomenclature
- naming or labelling of these groups to
distinguish one from the other
DEFINITIONS
◆ Identification
- assigning a name to an unknown by
comparison to characteristics of known
organism in a previously made classification
system
HISTORY of CLASSIFICATION
◆ 1700 Carl Linnaeus divided all living organisms into TWO

KINGDOMS
▼ Plantae and Animalia
▼ no allowance for single celled organisms such as
bacteria, fungi, algae
◆ 1866 Ernst Haeckel proposed THREE
▼ Plantae, Animalia and Protista
◆ 1966 Whittaker proposed FIVE KINGDOMS
▼ Plantae, Animalia, Protista, Fungi, Monera
CLASSIFICATION
◆ A classification system divides a larger group

into 2 or more smaller groups
▼ Hierarchical type classification tree
Enterobacteriaceae
 
Escherichia Klebsiella
   
E coli E hermanii K pneumoniae K oxytoca

KEY ELEMENTS OF
CLASSIFICATION SYSTEMS
◆ Morphological (phenotaxonomic)
◆ Physiological (phenotaxonomic)
◆ Cultural
◆ Biochemical (chemotaxonomic)
◆ Chemical
◆ Genetic (genotypic)
KEY ELEMENTS OF
CLASSIFICATION SYSTEMS
Morphological (phenotaxonomic)
Physiological (phenotaxonomic)
Cultural
Biochemical (chemotaxonomic)
Chemical
Genetic (genotypic)
Technolo
gy
MORPHOLOGICAL FEATURES
◆ Shape and size

◆ Cellular arrangement
◆ Motility
▼ number and distribution of flagella
◆ Endospore production
▼ size, shape, location
◆ Capsule
◆ Staining properties
PHYSIOLOGICAL GROWTH
REQUIREMENTS
◆ Oxygen/CO2 requirements
▼ aerobes, anaerobes, microaerophils
▼ obligate, facultative
◆ Temperature
▼ psychrophilic, mesophilic, thermophilic
▼ minimum, maximum, optimal
◆ Specialised nutritional growth factors
CULTURAL APPEARANCE
◆ Colonial morphology
▼ size and shape of colony (umbonate, rhizoid,
raised, swarming)
◆ Growth in liquid media
▼ surface, puffball
◆ Pigment production
◆ Extracellular products
▼ haemolysins, slime, lipases
BIOCHEMICAL ACTIVITY
(Phenotypic Characteristics)
◆ Respiratory Function
▼ catalase, oxidase, nitrate reduction
◆ Carbohydrate (CHO) utilisation
▼ method (O/F) and range of CHO
◆ Activity on nitrogenous compounds
▼ decarboxylation, urease, deaminases
◆ Hydrolysis of complex biochemicals
▼ proteases, lipases, DNAase
Minaturised
biochemical
from a single
colony
Rapid pre-
formed
enzyme
detection - 4
hours
Limitations
◆ API20E/Commercial Systems
▼ same biochemical substrates 1975-2003
▼ best test not on panel ➪ misidentification
▼ new organisms “need to fit” existing databases
▼ work well where large number of strains can be
compared ➪ accurate database
CHEMICAL COMPOSITION
◆ Cell wall amino acids

▼ gram positive genera
◆ Cell wall sugars
▼ speciation within species (ß haem Strep)
◆ Lipids - mycolic acids
▼ Mycobacterium, Nocardia
◆ Proteins
▼ electrophoretic patterns species specific
CHEMICAL COMPOSITION
◆ Problems
▼ Labour intensive
▼ Time consuming
▼ Technically demanding
▼ Relevant to select groups of organisms only
GENETIC CHARACTERISTICS
(genotype)
◆ Genome size
◆ G+C Ratio (1960s)
▼ constant within species but not unique
◆ Whole genome DNA-DNA homology (Brenner)
◆ 16sRNA (mid 1990s)
◆ Sequencing DNA (automated PRISM)
G+C RATIOS
◆ G+C Ratio
▼ bacterial genome is dsDNA (circular)
▼ G+C ratio determines the Tm
denaturation temperature
dsDNA➪ssDNA
▼ high G+C ➪ high Tm
▼ determined by HPLC of DNA hydrolysed by nuclease
P1
▼ G+C ratio constant for a particular species
▼ Range 24-76% (3% within species, 10% within
genus)
▼ different genera may have same G+C
DNA-DNA hybridization (1)
◆ total genome comparison

◆ measures the degree of DNA similarity
◆ variety of methods available
▼ labelled DNA - radioactive, biotin
▼ thermal stability of annealed strands
◆ >70% relatedness ➪ same species
▼ values vary with method (55-75% range)
▼ expensive, labour intensive, technically complex
DNA-DNA hybridization (2)
◆ problems
▼ many bacteria have sequences in common
▼ annealing of strands when up to 15% base
sequence is different
▼ Escherichia coli versus Shigella sp
◆ additional criterion - ▲Tm <5oC
▼ difference between Tm of known:known strain
against Tm of unknown:known
◆ International definition of genomospecies
Ribosomal RNA
◆ All living cell contain rRNA (highly conserved)
◆ bacteria possess
▼ 16S, 23S and 5S genes (1-11 copies)
◆ most useful method is rRNA sequencing
◆ 16S rRNA best due to size
▼ 1,500 base that can be sequenced in 1 day
▼ 23S (3,000), 5S (150)
◆ International databases GENBANK
◆ Useful for non-cultivatable bacteria
SEQUENCING
◆ Amplification of 16S RNA gene by PCR
◆ Sequence PCR product (ABI PRISM)
▼ ss DNA add primer + DNA polymerase
▼ add deoxyribonucleotides (dA,dT,dC,dG)
▼ add labelled didoxyribonucleotides individually
(limit one eg ddA)
▼ reaction stops when ddA are incorporated
▼ separate on gel
▼ determine sequence from position of band
Limitation of Molecular
Characterisation
◆ Survey only small part of genome
◆ Ribosomal genes highly conserved
▼ level of similarity required for species 97%
▼ level of difference strain not species
◆ Databases derived from 1-2 strains
▼ not reflect diversity of species
▼ related to biochemical differences (laboratory
level detection)
Evolution of Classification
Systems
◆ Pre 1900s - based on disease process
◆ Morphology, size, motility (Cohn)
◆ In vitro agar culture ➪ first biochemical tests
◆ 1920s - first heirarchial systems
▼ Bergey 1923, Topley and Wilson 1929
◆ Limited test types ➪ duplication of names
▼ using only a small number of easily measured tests
▼ different tests given more weight than others
Exercise 1
Shape
Colour - yellow
NUMERICAL TAXONOMY (1)
◆ Computer analysis proposed by Sneath 1959
◆ Principle - all phenotypic characters are weighted equally
◆ Uses 100 or more phenotypic characteristics
◆ 300-400 organisms compared simultaneously
◆ Entered into computer database as + or -
◆ Determine the coefficient of similarity
◆ Develop dendogram (phenogram)
▼ joins individual strains into groups
NUMERICAL TAXONOMY (2)
◆ Problems
▼ what % similarity = genus (70%)
▼ what % similarity = species (80%)
▼ what type of tests to include
phenotypic diversity ✖ genotypic
diversity
▼ how many of each different test type
▼ is a negative a true negative
EXERCISE 2
A
B
C
D
E
60 70 80 90
Polyphasic Species Concept
◆ Developed by Colwell 1970
◆ Integrates all available phenotypic, genotypic and
phylogenetic information to achieve a classification
scheme that facilitates identification
e.g. Bordetella
- 3 species (pertussis, parapertussis, bronchiseptica)
- all > 80% DNA homology (?single species)
- are 3 distinct species as they differ in numerous phenotypic and
chemotaxonomic tests.
NOMENCLATURE
◆ System of naming organisms

◆ Name defines the organism without listing
individual characteristics
◆ Names traditionally taken from Latin
◆ Binomial
▼ genus with a capital letter
▼ species with no capital letter
◆ Italicised or underlined
International Codes of
Nomenclature
◆ International Code for naming organism
▼ must be fully characterised (pheno/geno)
▼ must be a type strain in recognised collection -
NCTC, ATCC and UQCC
▼ “priority of publication” applies
◆ Approved List of Bacterial Names 1980
◆ Valid names published annually in IJSB
▼ only recognised publication
Bacteriological Code (1990)
◆ Lists the rules of bacterial nomenclature
▼ outline rules for generation of new names to
ensure continuity between disciplines
▼ avoid names that create error of confusion
▼ revision (new species 5-10 strains from
geographically unrelated areas)
▼ revision (new species independently confirmed)
▼ set procedures for handling disputes
◆ ICBS Judicial Committee presides over disputes
Why Names Change?
◆ New genetic data
▼ Pseudomonas has 5 distinct groups
▼ New genera created (Burkholderia, Comamonas,
Acidovorax, Brevundimonas, Stenotrophomonas)
◆ Single strain genera
▼ genetic and phenotypic variation from other
known genera
▼ e.g. Stenotrophomonas (formerly Pseudomonas,
Xanthomonas)
Identification Systems
◆ Practical application of taxonomy

◆ Evolution of ID systems
▼ Dichotomous Key
▼ Flowchart
▼ Diagnostic Tables
▼ Probability Tables
Keys and Flowcharts
◆ Keys use a series of characters in turn

◆ Limitations
▼ assumes all strains will be either positive or
negative
◆ Flowcharts use a series of characters in a set
order
▼ allow for some variation within species
▼ species appear more than once in the chart
Diagnostic Table
◆ list a series of characters in any order

◆ each result is categorised
▼ + = >85% positive
▼ V = 16-84% positive
▼ w = weak reaction
▼ () = delayed reaction
◆ time consuming
◆ manual application only
Probability Tables
◆ % positivity determined for each test

◆ ideal for computerisation
◆ provides for % certainty of ID
◆ limitations
▼ test large numbers of same species
▼ test from different geographical locations
▼ test human, veterinary and environmental
▼ needs to be an ongoing process
Ideal Identification System
◆ Combination
▼ phenotypic, chemotaxonomic, molecular
◆ Not practical for all situations
▼ New species ➪ ALL
▼ Clinical laboratory ➪ phenotypic, chemo
▼ Special situations ➪ molecular backup
◆ Evolution!

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Taxonomy,

◆ Be able to define and explain differences between

“Taxonomy is written by taxonomists in this form the

It is the most subjective branch of biological science

◆ 1700 Carl Linnaeus divided all living organisms into TWO

◆ A classification system divides a larger group

E coli E hermanii K pneumoniae K oxytoca

◆ Shape and size

◆ Cell wall amino acids

◆ total genome comparison

◆ System of naming organisms

◆ Practical application of taxonomy

◆ Keys use a series of characters in turn

◆ list a series of characters in any order

◆ % positivity determined for each test

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