Identifying and Classifying Microorganisms

Chapter 10: Identifying and Classifying Microorganisms

A Glimpse of History

  • Cohn: Initial classification of bacteria based on shape in the 1870s.

  • Orla-Jensen: Classification by physiology introduced in 1908.

  • Kluyver and van Niel: Evolutionary relationship classification in the 1930s.

  • Stanier: Classification based on nucleic acid sequences in 1970.

  • Woese: Division of prokaryotes into two major groups based on ribosomal RNA sequences in the late 1970s. This led to the three-domain system:

    • Bacteria

    • Archaea

    • Eukarya

Principles of Taxonomy

  • Definition: Taxonomy is the science that characterizes and names organisms, arranging them into hierarchical groups (taxa).

  • Three areas of taxonomy:

    • Identification: Characterizing organisms for grouping.

    • Classification: Arranging organisms into related groups.

    • Nomenclature: System of assigning names to organisms.

Strategies for Identifying Microorganisms

  • Methods for identifying microorganisms include:

    • Microscopic examination

    • Culture characteristics

    • Biochemical tests

    • Nucleic acid analysis

    • Patient symptoms for pathogens

  • Identification is often prioritized over determining evolutionary relationships.

  • Point-of-care testing (POCT): Testing performed at or near patient care sites.

Strategies for Classifying Microorganisms

  • Phylogeny: Considers evolutionary relatedness.

  • Species: Basic unit in prokaryotes, defined as a group of closely related isolates or strains.

  • Challenges:

    • Difficulty in determining distinct species or strains within a species.

    • Subspecies divisions exist.

  • Informal groupings used:

    • Lactic acid bacteria

    • Anoxygenic phototrophs

    • Endospore-formers

    • Sulfate reducers

Taxonomic Hierarchies

  • Species: A group of closely related strains or individuals.

  • Genus: A collection of similar species.

  • Family: A collection of similar genera. Nomenclature ends with -aceae.

  • Order: A collection of similar families. Nomenclature ends with -ales.

  • Class: A collection of similar orders, usually named with -ia.

  • Phylum (sometimes division): A collection of similar classes, ending in -ota.

  • Kingdom: A collection of similar phyla or divisions.

  • Domain: A collection of similar kingdoms, reflecting the cell characteristics of organisms.

Current Classification System

  • Three-domain system based on nucleotide sequences in rRNA, replacing Whittaker’s five-kingdom system from 1969.

  • Expanded using amino acid sequences of ribosomal proteins, showing many unculturable bacteria.

Comparison of Properties in the Three Domains

Cell Feature

Archaea

Bacteria

Eukarya

Cytoplasmic membrane lipids

Hydrocarbons linked by ether linkage

Fatty acids linked by ester linkage

Fatty acids linked by ester linkage

Membrane-bound nucleus

No

No

Yes

Peptidoglycan cell wall

No

Yes

No

Presence of introns

Sometimes

No

Yes

Ribosomes

70S

70S

80S

Bergey’s Manual of Systematic Bacteriology

  • Describes all known prokaryotic species.

  • Newest edition consists of five volumes.

  • Classifies organisms based on genetic relatedness (phylogeny), ecology, culture methods, enrichment, isolation, and maintenance methods.

Identification Methods Based on Phenotype

  • Microscopic morphology is the crucial initial step for determining size, shape, and staining characteristics.

  • Gram stain distinguishes between Gram-positive and Gram-negative bacteria, which may guide initial therapy.

  • Special stains can aid in identifications, such as the acid-fast stain for identifying Mycobacterium tuberculosis.

Culture Characteristics

  • Characteristics of cultures can reveal identity, such as:

    • Serratia marcescens colonies appearing red at 22°C.

    • Pseudomonas aeruginosa producing green pigment with a distinct fruity odor.

  • Use of differential media aids in identification:

    • Streptococcus pyogenes yields β-hemolytic colonies on blood agar.

    • E. coli ferments lactose, forming pink colonies on MacConkey agar.

Metabolic Capabilities Revealed by Biochemical Tests

  • Catalase Test: Positive if bubbles appear upon adding hydrogen peroxide (H₂O₂) to a colony.

  • Many biochemical tests rely on pH indicators.

  • Sugar fermentation lowers pH, possibly trapping gas in inverted tubes. Conversely, urease production raises pH.

Biochemical Tests Strategy

  • Typical identification uses dichotomous keys for series of alternative choices to guide pathways through tests, speeding the process.

  • Some tests, such as breath tests for urease (e.g., Helicobacter pylori), can be accomplished without culturing.

Commercial Identification Kits

  • Rapid identification via biochemical tests, albeit requiring incubation periods. Results create patterns that a computer can compare against databases.

Serological Characteristics

  • Utilizes antibodies to detect specific molecules (e.g., proteins, polysaccharides).

  • Useful markers include: surface structures such as cell walls, capsules, flagella, and pili. Certain Streptococcus species contain unique carbohydrates in their walls.

Protein Profile Analysis

  • MALDI-TOF (matrix-assisted laser desorption ionization time of flight mass spectrometry) can rapidly determine protein profiles, often in less than 15 minutes.

    • Measures the mass of components via mass spectrometry, creating a mass spectrum profile for comparison against a database.

Identification Methods Based on Genotype

  • Tests detect sequences unique to specific species or groups through:

    • Nucleic acid probes

    • Nucleic acid amplification tests (NAATs)

  • Limitation: these tests detect only single possibilities, requiring multiple probes if testing diverse species.

Nucleic Acid Probes

  • Locate nucleotide sequences characteristic of species, demonstrated by first increasing the DNA in a sample for denaturation and hybridization with a labeled probe.

    • Fluorescence in situ hybridization (FISH) probes for 16S rRNA do not require amplification steps.

Nucleic Acid Amplification Tests (NAATs)

  • Increase copies of specific DNA sequences for detection even in limited quantities from various sources (e.g., body fluids, soil, etc.).

  • Commonly used technique: Polymerase chain reaction (PCR).

Sequencing Ribosomal RNA Genes

  • 16S rRNA sequences are stable and particularly useful for microbial identification due to moderate size (approximately 1,500 nucleotides). Comparisons with extensive databases can identify uncultivable organisms.

Characterizing Strain Differences

  • Important for foodborne illnesses, forensic investigations, and disease diagnosis. Strains can be characterized through:

    • Biochemical Typing: Groups with characteristic patterns known as biovars or biotypes.

    • Serological Typing: Distinguishing E. coli by its antigenic types, such as E. coli O157:H7 and other serovars.

Whole Genome Sequencing (WGS)

  • Modern DNA sequencing methods allow easy detection of subtle differences among strains that appear identical phenotypically. WGS has replaced historical methods based on restriction fragment polymorphisms (RFLPs).

  • WGS is now used for tracking infectious disease outbreaks by various national and global surveillance networks.

WGS Data Networks

  • PulseNet: A CDC network tracking foodborne outbreaks via WGS data from laboratories.

  • Genome Trakr: An FDA-associated network collecting WGS data for foodborne pathogen tracking.

  • Programs like Nextstrain utilize WGS data to track viral pathogen evolution and spread across populations.

Phage Typing

  • Phage typing identifies bacterial strains based on their susceptibility to specific bacteriophages, useful in labs lacking modern molecular typing equipment.

Antibiograms

  • Reveal differences in bacterial susceptibility to antimicrobial medications, where clear zones indicate susceptibility around antimicrobial discs on agar plates.

Classifying Microorganisms

  • Classification has historically been phenotype-based, including size, shape, staining, and metabolic capabilities.

  • New molecular techniques have improved accuracy in determining relatedness and constructing phylogenetic trees based on random mutations.

Sequence Analysis of Ribosomal Components

  • Ribosomes serve crucial and constant functions, making ribosomal RNA comparisons reliable for classification purposes. Limited mutations are tolerable, preserving sequence similarities across distantly related organisms.

DNA-DNA Hybridization (DDH)

  • DDH assesses relatedness by measuring similarity in nucleotide sequences via hybridization rates between single-stranded DNA. Over 70% similarity indicates the same species. For instance, Shigella and Escherichia may be classified as the same species.

Genome Sequence Analysis

  • Current trends emphasize WGS over DDH for evaluating organism relatedness by comparing shared genes and calculating the average nucleotide identity (ANI).

G + C Content

  • The percentage of G-C base pairs influences genetic relationship assessments; a difference of several percentages suggests unrelatedness. Higher G-C content DNA melts at higher temperatures due to stronger hydrogen bonding.

Conclusion

  • Phenotypic methods, while historically significant, have largely been supplanted by DNA sequence data. Taxonomists emphasize the importance of multiple dimensions (phenotypic and genotypic) for effective microbial classification and identification.

  • Identification Methods Based on Phenotype: Techniques to identify microorganisms through observable characteristics.

    • Microscopic morphology: Analyzing size, shape, and arrangement through microscopy.

    • Gram stain: A staining technique that differentiates bacteria based on their cell wall composition into Gram-positive and Gram-negative.

    • Special stains: Additional stains like the acid-fast stain aid in identifying specific bacteria (e.g., Mycobacterium tuberculosis).

  • Culture Characteristics: Observations made from colony characteristics in culture.

    • Colony appearance at different temperatures: Color or morphology of colonies at varying temperatures can be indicative of specific species.

    • Differential media usage: Growth media designed to distinguish different organisms based on their biochemical characteristics (e.g., E. coli on MacConkey agar).

  • Metabolic Capabilities Revealed by Biochemical Tests: Assessing biochemical properties of microorganisms.

    • Catalase test: Tests for the enzyme catalase by observing if bubbles form when hydrogen peroxide is applied to a colony.

    • pH indicators: Used to detect changes in pH resulting from metabolic activities (sugar fermentation lowers pH, urease production raises it).

  • Commercial Identification Kits: Ready-to-use kits that provide rapid identification based on biochemical tests, usually requiring incubation for accurate results.

  • Serological Characteristics: Techniques utilizing antibodies to recognize specific antigens on the microorganism's surface, providing clues for identification.

  • Protein Profile Analysis: A method using MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrometry to measure protein profiles for identification.

  • Identification Methods Based on Genotype: Techniques that identify organisms based on their genetic material.

    • Nucleic acid probes: Specific sequences in the DNA that can be detected to identify particular species.

    • Nucleic acid amplification tests (NAATs): Techniques that amplify DNA to detect presence even in small quantities (e.g., PCR).

  • Sequencing Ribosomal RNA Genes: Analyzing sequences of rRNA genes (such as 16S rRNA) for microbial identification due to their conserved nature.

  • Characterizing Strain Differences: Methods used to distinguish between strains within a species.

    • Biochemical typing: Groups organisms based on distinct biochemical patterns known as biovars or biotypes.

    • Serological typing: Identifying strains based on their specific antigenic types.

  • Whole Genome Sequencing (WGS): A modern technique that sequences the entire genome, allowing for detection of minor differences among strains that appear similar.

  • Phage Typing: A method of identifying bacterial strains based on their susceptibility to specific bacteriophages (viruses that infect bacteria).

  • Antibiograms: Tests that reveal differences in bacterial susceptibility to various antimicrobial agents by observing clear zones around antibiotic discs on agar plates.

  • Microorganisms: Tiny living organisms, visible only through a microscope, including bacteria, viruses, fungi, and protozoa.

  • Unculturable Bacteria: Bacteria that cannot be grown in standard laboratory culture conditions.

  • Differential Media: Growth media designed to distinguish different organisms based on their biochemical characteristics.

  • Acid-fast Stain: A special stain used to identify Mycobacterium tuberculosis.

  • Biochemical Typing: Groups organisms based on distinct biochemical patterns known as biovars or biotypes.

  • Dichotomous Keys: A flowchart-like tool used for identifying organisms based on a series of choices between alternative characteristics.

  • Mass Spectrometry: An analytical technique used to measure the mass-to-charge ratio of ions for molecular identification.

  • pH Indicators: Chemicals that change color in response to the acidity or alkalinity of a solution, used in various biochemical tests.

  • Amplification: The process of increasing the number of copies of a specific DNA sequence to improve detection.

  • Hybridization: A process used in nucleic acid probes to allow complementary DNA strands to bind together, indicating the presence of a target sequence.

  • PulseNet: A CDC network tracking foodborne outbreaks using whole genome sequencing data.

  • Genome Trakr: An FDA-associated network collecting whole genome sequencing data for tracking foodborne pathogens.

  • Nextstrain: A program that utilizes whole genome sequencing data to track viral pathogen evolution and spread.

  • Enrichment Cultures: Special culture conditions that allow the growth of a particular microorganism while inhibiting others, enhancing identification chances.

  • Antimicrobial Susceptibility Testing: One of the procedures performed to determine the effectiveness of antibiotics against specific microorganisms.

  • Filtration: A method to separate microorganisms from liquid samples through a filter with specific pore sizes.

  • Pathogens: Microorganisms that cause disease in a host organism.

  • Eukarya: One of the three domains of life, containing organisms with complex cells.

  • Archaea: One of the three domains of life, comprising prokaryotic organisms often found in extreme environments.

  • Bacteria: One of the three domains, including single-celled prokaryotic organisms.

  • Clinical Microbiology: The branch of microbiology that deals with the diagnosis and management of infectious diseases.

  • Phenotype: Observable physical or biochemical characteristics of an organism, determined by genetic expression.

  • Genotype: The genetic constitution of an organism.

  • Cloning: The process of creating identical copies of a biological entity.

  • Isolation: The process of separating a microorganism from a mixture to study or identify it individually.

  • Ribosomal RNA (rRNA): RNA components of the ribosome, essential for protein synthesis in all living cells.

  • Mutation: A change in the DNA sequence of an organism that may lead to changes in traits or characteristics.

  • Antisera: Serum that contains antibodies against specific antigens, often used in serological tests.

  • Taxonomy: The science that characterizes and names organisms, arranging them into hierarchical groups (taxa).

  • Identification: Characterizing organisms for grouping.

  • Classification: Arranging organisms into related groups.

  • Nomenclature: System of assigning names to organisms.

  • Phylogeny: Considers evolutionary relatedness.

  • Species: Basic unit in prokaryotes, defined as a group of closely related isolates or strains.

  • Genus: A collection of similar species.

  • Family: A collection of similar genera, nomenclature ends with -aceae.

  • Order: A collection of similar families, nomenclature ends with -ales.

  • Class: A collection of similar orders, usually named with -ia.

  • Phylum: A collection of similar classes, ending in -ota.

  • Kingdom: A collection of similar phyla.

  • Domain: A collection of similar kingdoms.

  • Three-domain system: Classification system based on nucleotide sequences in rRNA, replacing Whittaker’s five-kingdom system.

  • Bergey’s Manual of Systematic Bacteriology: Describes all known prokaryotic species and classifies organisms based on genetic relatedness, ecology, culture methods, and maintenance methods.

  • Gram Stain: A staining technique that distinguishes between Gram-positive and Gram-negative bacteria.

  • Catalase Test: Tests for the enzyme catalase by observing if bubbles appear upon adding hydrogen peroxide to a colony.

  • Serological Characteristics: Techniques utilizing antibodies to recognize specific antigens on the microorganism's surface.

  • MALDI-TOF: A method using mass spectrometry to measure protein profiles for identification.

  • Nucleic Acid Probes: Locate nucleotide sequences characteristic of species.

  • Nucleic Acid Amplification Tests (NAATs): Techniques that amplify DNA to detect presence even in small quantities.

  • Whole Genome Sequencing (WGS): A technique that sequences the entire genome, allowing for detection of minor differences among strains.

  • Phage Typing: A method of identifying bacterial strains based on their susceptibility to specific bacteriophages.

  • Antibiograms: Tests that reveal differences in bacterial susceptibility to various antimicrobial agents by observing zones around antibiotic discs on agar plates.