Chapter9 Microbial taxonomy

Three separate but interrelated parts: 1.

Classification: the arrangement of organisms into groups or taxa based on mutual similarity or evolutionary relatedness.

2.

3.

Nomenclature : the branch of taxonomy concerned with the assignment of names to taxonomic groups in agreement with published rules.

Identification: the practical side of taxonomy, the process of determining that a particular isolate belongs to a recognized taxon.

Taxonomy is important for several reasons

1.

2.

3.

4.

It allows us to organize huge amounts of knowledge about organisms Allows us to make predictions and frame hypotheses for further research based on knowledge of similar organisms.

It places microorganisms in meaningful, useful groups with precise names so that microbiologists can work with them and communicate efficiently.

Identification of microorganisms accurately

Taxomomic ranks

      Strain: one single isolate or line Type: sub-set of species Species: related strains Genus: related species Family: related genera Order; class; phylum; domain

Related concepts

A species: a collection of strains that have a similar G+C composotion and 70% or greater similarity as judged by DNA hybridization experiments.

A biovars: variant procaryotic strains characterized by biochemical or physiological differences.

Morphovars: differ morphologically Serovars: have distinctive antigenic properties Type strain: it is usually one of the first strains studied and often is more fully characterized than other strains

Classification systems

Phenetic classification: one that groups organisms together based on the mutual similarity of their phenotypic characteristics. Comparing as many attributes as possible.

Numerical taxonomy : computers may be used to analyze data for the production of phenetic classification.

Information about the properties of organisms is converted into a form suitable for numerical analysis and then compared by means of a computer.

Phylogenetic classification : based on evolutionary relationships rather than general resemblance.

defficult because of the lack of a good fossil record. Comparision of genetic material and gene products

Major characteristics used in taxonomy

Morphological characteristics Physiological and metabolic characteristics Ecological characteristics Genetic analysis Molecular characteristics

Morphological characteristics

Cell shape Cell size Cilia and flagella Cellular inclusions Color Mechanism of motility Endospore shape and location Spore morphology and location Colonial morphology Ultrastructural characteristics Staining behavior

Physiological, metabolic and Ecological characteristics

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Carbon and nitrogen sources Cell wall constituents Energy sources Fermentation products Luminescence Motility Osmotic tolerance Storage inclusions

       

General nutritional type Growth temperature optimum and range Mechanisms of energy conversion pH optimum and growth range Photosynthetic pigments Salt requirements and tolerance Secondary metabolites formed Sensitivity to metabolic inhibitors and antibiotics

Genetic analysis

The study of transformation and conjugation in bacteria is sometimes taxonomically useful. Plasmid-borne traits can cause errors in bacterial taxonomy if care is not taken.

Transformation can occur between different procaryotic species but only rarely between genera.

E.coli can undergo conjugation with the genera Salmonella and Shigella but not with Proteus and Enterobacter

Molecular analysis

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Historical guanine (G)+ cytosine (C) (% GC)

• •

Now Hybridization Gene characterization

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sequencing

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other

DNA-DNA hybridization

Heat Strain 1 + 0% Homology Strain 2 100% Homology

DNA-DNA hybridization

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Groups bacterial strains into species

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Below species level

•

little or no relatedness

16S rRNA Sequencing

 similarity above species level  allows relatedness comparisons of all bacteria  closely related bacterial species may be identical  development of clinical tests based on sequence

Ribosomal RNAs as Evolutionary Chronometers



Reasons:

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Ancient molecules

• •

Functionally constant Universally distributed

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Moderately well conserved in sequence across broad phylogenetic distances

The 16S rRNA or 18S rRNA Technique

The 16S rRNA or 18S rRNA Technique

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In Prokaryotes:

• • •

5S rRNA is too small, contains limited info 23S rRNA is too large, too difficult to manage 16S rRNA has the right size for studies



In Eukaryotes:

•

18S rRNA is used for phylogenetic measurements

The 16S rRNA or 18S rRNA Technique

The 16S rRNA or 18S rRNA Technique

 Ribosomal Database Project (RDP):  • http://www.cme.msu.edu/RDP Compare your sequences with the database to find out the organisms you identify

Phylogenetic Trees from DNA Sequences 

Distance-Matrix Method for generating the trees

 

Evolutionary Distance (E D ) Computer compare the sequence differences and build the phylogenetic tree based on corrected E D

Phylogenetic Trees from DNA Sequences

Signature Sequences: unique to certain group of organisms Applications: Phylogenetic Probes

“official” taxonomy and nomenclature

  Taxonomy: Bergey’s manual of systematic bacteriology or Bergey’s manual of

determinative bacteriology

in some sence official Nomenclature: all new names are validly published to gain standing in the nomenclature, either by being published in papers in the international Journal of Systematic Bacteriology or, if published elsewhere, by being announced in the Validation Lists

Bergey’s mamual of systematic bacteriology

The first edition: phenetic Procaryotic groups are divided into four volumes: (1)gram-negative bacteria of general, medical, or industrial importance.

(2)gram-positive bacteria other than actinomycetes (3)gram-negative bacteria with distinctive properties, cyanobacteria, and archaea (4)actinomycetes

Bergey’s mamual of systematic bacteriology

The second edition: phylogenetic Five volumes: Volume 1: the archaea, and the deeply branching and phototrophic bacteria Volume 2: the Proteobacteria Volume 3: the low G+C gram-positive bacteria Volume 4: the high G+C gram-positive bacteria Volume 5: the Planctomycetes, Spirochaetes, Fibrobacteres, Bacteroidetes, and Fusobacteria

Table19.9 p441

Table19.8 p436

Identification in the diagnostic laboratory

• • • •

Aids treatment Susceptibility – antibiotic selection Based on taxonomy Simple, low cost, rapid

Steps in isolation and identification

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Step 1. Streaking culture plates

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colonies on incubation (e.g 24 hr)

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size, texture, color, hemolysis oxygen requirement

Blood Agar Plate

Isolation and identification

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Step 2. Colonies Gram stained

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cells observed microscopically

Gram negative Heat/Dry Crystal violet stain Gram positive Iodine Fix Alcohol de

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stain Safranin stain

Step 3. Isolated bacteria are speciated



Generally using physiological tests Typical Culture Laboratory Bench

Step 4

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Antibiotic susceptibility testing

Susceptible Not susceptible Bacterial lawn No growth Growth Antibiotic disk

Rapid diagnosis without culture

• • •

WHEN AND WHY?

grow poorly

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Isolation slow may not be clinically useful can not be cultured

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isolation impossible

Rapid

“

Strep

”

Streptococcal antigenic extract

Test

Antibody Latex beads

Bacterial DNA sequences amplified directly from human body fluids

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Polymerase chain reaction (PCR)

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Great success in rapid diagnosis of tuberculosis.

Microscopy

• •

spinal fluids (meningitis

脑膜炎

) sputum (tuberculosis)

•

sensitivity poor

Serologic identification

• antibody response to the infecting agent • several weeks after an infection has occurred

Questions

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6.

7.

Why is Ribosomal RNAs used as Evolutionary Chronometers Why is 16S rRNA employed to study phylogenetics rather than the smaller 5S rRNR and the large 23S rRNA in Prokaryotics?

How to sequence 16S rRNA from inside the cells?

How to identify a prokaryotic or eukaryotic organism based on 16S or 18S rRNA?

How to build Phylogenetic Trees from DNA Sequences?

Why it is said the archaea closer to eukarya than bacteria is?

What is signature sequence and how can it be used?