10 Identifying and Classifying Microorganisms

Taxonomy

Science of classifying organisms into taxa (groups)

Divided into groups based on phenotypic and genotypic relatedness

Identification

Process of characterizing in order to group

Classification

Arranging organisms into similar or related groups

Nomenclature

System of assigning names. Provides universal names for organisms

Taxonomic Hierarchies

[__]: classification categories arranged in hierarchical order.

From the most basic unit to the most complex:

Species » a group of closely related strains or individuals

(different definitions under different domains)

(Eukaryotes) group of organisms capable of producing fertile offspring

(Prokaryotes) group of closely related isolates or strains: genetic variant or subtype of a species

Genus » a collection of similar species

Family » a collection of similar genera. Suffix -aceae

Order » a collection of similar families. Suffix -ales

Class » a collection of similar orders. Suffix -ia

Phylum » “division.” A collection of similar classes. Sufffix -ota

Kingdom » a collection of similar phyla or divisions

Domain » 3 » Eukarya, Bacteria, Archaea

Phylogeny

Evolutionary relatedness

A three-domain system based on rRNA sequence by Carl Woese et. al; how close they are show how related they are

Fun fact: Eukarya are more closely related with Archaea than Archaea are with Bacteria

a. based on ribosomal RNA data

b. based on ribosomal protein sequence data

morphology

Classification was historically based on physiology (phenotype) and

[__]: size, shape, staining characteristics, and metabolic capabilities

Current techniques are more accurate with molecular techniques

• comparison of molecular sequences of DNA, RNA, and protein

• accurate construction of phylogenetic tree: tree that shows divergence and relationships of organisms

• transfer mechanism of prokaryotes

  • horizontal and lateral transfer of DNA

microscopic morphology

Method used to identify microorganisms. Involve analysis of size, shape, and staining characteristics; looking at things under the microscope

eg.

• Gram stain: distinguishes between Gram-positive (purple) and Gram-negative bacteria (pink)

  • may provide sufficient information to start appropriate therapy

• Special stains → useful providing additional info (acid-fast, endospore, capsule)

Can diagnose infections:

Culture Characteristics

Method used to identify microorganisms.

Growing bacteria/culturing them to see their colony morphology, or [__] → gives initial clues to the identity of an organism

• Serratia marcescens has red pigment at 22C

• Pseudomonas aeruginosa produces green pigment and a distinct fruity odor

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Differential media: culture media that contains a substance that can be changed by a specific bacteria, helps as well

• Streptococcus pyogenes » strep throat. has beta-hemolytic colonies on blood agar

• E.coli » urinary tract infection. ferments lactose, creating pink colonies on MacConkey agar

Metabolic capabilities

Method used to identify microorganisms.

A series of biochemical tests performed to identify the organism.

Identifies the presence of enzymes

• Catalase test: detect if organism produces catalase to protect against hydrogen peroxide

  • adding hydrogen peroxide and formation of O₂ and water = catalase-positive

• pH indicators

  • Sugar Fermentation: detects fermentation of sugar and lowers pH if so

    • yellow - positive

    • red - negative

    • orange - control / uninoculated

  • Urease Production: detects urease (enzyme that degrades urea to CO₂ and ammonia)

    • pink = positive

    • yellow = control / un-inoculated

When commercialized, allow for rapid identification

• can run series of test with single inoculation

• standardized and uniform tests are easier for comparison

• enterotube → each chamber has 1-2 tests. single needle can inoculate all of the chambers = multiple test results

Dichotomous key

Flowchart of tests with positive and negative results.

Used to identify bacteria by asking successive questions that relate to morphological or biochemical characteristics

Last question should differentiate between two species

Serology

Method used to identify microorganisms.

Uses antibodies to detect and identify antigens found on proteins and polysaccharides that make up a microorganism.

• since specific antigens stimulate specific antibodies, it relies on that specific interaction to identify an organism

• commercial antibodies available for specific bacteria → rapid detection of organism

eg. Streptococcus pyogenes - causative agent of strep throat

Serological typing

Uses antibodies to identify antigen differences among strains as distinguishing markers

eg. → E.coli O157:H7

E.coli distinguished by antigenic type of flagella (H antigen), capsules, and lipopolysaccharide (O antigen)

leads to groupings like serovar or serotype

Serovar: group of strains that have a characteristic antigenic structure that differs from other strains

Genotyping

Determines nucleotide sequences

• identifies species or group based on unique sequences

• identifies organisms that cannot be grown in culture

Sequencing RNA Genes

The amplification and sequencing of 16S (prokaryotes) / 18S (eukaryotes) rRNA

This is due to the little genetic variation in rRNA (highly preserved)

→ can compare sequence to database to show the nearest relative and what organism it is

→ can identify organisms that cannot be grown in culture

Nucleic acid amplification tests

also known as NAATs, they amplify specific sequences for detection and or identification of

1. small numbers of organisms (eg. organisms in body fluids, soil, food, water)

2. organisms that grow slowly

→ eg. Mycobacterium tuberculosis

» much faster (3-4h vs. 6 weeks for cultures)

3. organisms that cannot be cultured

Polymerase chain reaction (PCR) is a common technique

Molecular Typing

Uses differences in DNA sequences to distinguish phenotypically identical strains

Used to trace epidemics and outbreaks

eg. DNA fingerprinting

• cut all DNA samples with the same restriction enzyme

• separate via gel electrophoresis » bands migrate according to size

• use restriction fragment length polymorphisms (RFLPs): method that compares patterns of DNA fragment sizes, to identify

• different RFLPs = different strains

Antibiograms

Identifies organisms based on antibiotic susceptibility patterns; used to distinguish between different bacterial stains

• disc impregnated with antimicrobials placed on inoculated plate

• zone of inhibition → area that clears. we look at this for susceptibility patterns

  • (if low) tells microbial susceptibility » lack of ability to resist microbial infections/disposition to disease

• different strains = different susceptibility patterns