Chapter 10: Identifying & Classifying Microorganisms

Species

A group of closely related strains or individuals

Genus

A collection of similar species

Family

A collection of similar genera. In prokaryotic nomenclature, the name of the family ends in the suffix -aceae

Order

A collection of similar families. In prokaryotic nomenclature, the name of the order ends in the suffix -ales

Class

A collection of similar orders

Phylum

(sometimes called a division) A collection of similar classes

Kingdom

A collection of similar phyla or division

Domain

A collection of similar kingdoms. The domain reflects the characteristics of the cells that make up the organism.

Classification

The process of arranging organisms into similar or related groups (taxa), primarily to make it easier to identify them for study

Identification

The process of characterizing an isolate in order to determine the group (taxon) to which it belongs

Phylogeny

Evolutionary relatedness of organisms

Strain

A pure culture isolate; subgroup within a species

Taxonomy

The science of characterizing organisms to arrange them into hierarchical groups (taxa); involves three interrelated areas: identification, classification, and nomenclature

Ferdinand Cohn

Researched bacteria classified by shape

Orla-Jensen

Researched classification by physiology

Kluyver and van Niel

Researched classification based on evolutionary relationships

Stanier

Researched relationships determined by comparing physical traits, nucleotide sequences

Woese

Researched prokaryotes divided into two major groups based upon ribosomal RNA sequences; led to the modern three-domain classification system

Microorganism Identification Strategies

Microscopic examination, culture characteristics, biochemical tests, nucleic acid analysis, and patient symptoms (for pathogens)

Cytoplasmic membrane lipids (archaea)

Hydrocarbons (not fatty acids) linked to glycerol by ether linkage

Cytoplasmic membrane lipids (bacteria and eukarya)

Fatty acids linked to glycerol by ester linkage

Introns

Present in eukarya, sometimes present in archaea, and not present in bacteria

Bergey’s Manual of Systematic Bacteriology

The most important reference for taxonomic descriptions of bacteria; it includes information on methods for maintenance and preservation of organisms, classifies all known prokaryotes based on their phylogeny, and includes information on ecology, methods of enrichment, culture, and isolation of organisms

International Code of Nomenclature of Prokaryotes

Provides rules for naming bacteria and archaea; the three-domain system of classification places all life into three domains based upon ribosomal RNA nucleotide sequences; morphological features such as shape and size can be used to help with classification, but many bacteria are too similar

Microscopic morphology

A method used to identify microorganisms; utilizes size, shape, staining characteristics for identification; sometimes enough to diagnose eukaryotic infections; requires further testing 

Gram-staining

A stain that distinguishes between Gram-positive and Gram-negative bacteria; results may be enough to start appropriate therapy

Acid-fast stain

A stain that may help identify Mycobacterium tuberculosis

Culture characteristics 

A method used to identify microorganisms; colony morphology that can give clues to the identity of the organism; for example, Serratia marcescens colonies are often red at 22 degrees Celsius, and Pseudomonas aeruginosa often produces green pigment; cultures also have a distinct fruity odor

Differential media

Media that aids in identification based on how it appears on different mediums; for example, Streptococcus pyogenes (strep throat) yields β-hemolytic colonies on blood agar and E. coli (urinary tract infection) ferments lactose, forms pink colonies on MacConkey agar

Metabolic capability

A method used to identify microorganisms; determined by biochemical tests like the catalase test (which is positive if bubbles are seen after H2O2 is added to the colony) or pH indicators (Sugar fermentation lowers pH and may trap gas in inverted tube and Urease raises pH)

Dichotomous key

Series of alternative choices; simultaneous tests speed process and provide more conclusive results; some tests accomplished without culturing (breath test to assay urease and identify Helicobacter pylori)

Commercial Kits

Allow rapid identification via biochemical tests; require incubation period; pattern of results is scored and a computer identifies the organism

Point of care testing (POCT)

Diagnostic methods done at or near the site of patient care; features in which tests for pathogen identification can be done near the site of patient care 

Serological testing 

A method used to identify microorganisms that uses antibodies to detect specific molecules; proteins, polysaccharides of prokaryotic cells can serve as identifying markers; most useful include surface structures of cell wall, capsule, flagella, pili; some Streptococcus species contain unique carbohydrate in cell wall

Protein profile

A method used to identify microorganisms; information that can be determined by MALDI-TOF; the unique pattern or “fingerprint” of proteins and other macromolecules present in a cell; it represents the masses of various cellular components and can be used to identify an organism; typically displayed as a mass spectrum, which shows the distribution of protein ions based on their mass-to-charge ratio.

MALDI-TOF (Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry)

A rapid analytical technique used to determine an organism’s protein profile, often within 15 minutes; a sample mixed with a matrix is placed on a sample plate. A laser beam vaporizes and ionizes the sample, producing charged particles (ions); smaller ions travel faster to the detector than larger ones, allowing their mass-to-charge ratios (m/z) to be measured. The resulting mass spectrum acts as a molecular fingerprint of the organism; the computer compares this spectrum to a reference database to identify the microorganism.

Specific Nucleotide Sequences 

Detection of these is a method used to identify microorganisms; tests can identify sequences unique to species or groups even in small numbers; tests that can be used to detect these are nucleic acid probes and nucleic acid amplification tests (NAATs); a significant limitation is that each detects only a single possibility, making it necessary to run multiple probes if the organism being tested could be one of multiple different species or related groups; relies on a preliminary step to amplify DNA 

DNA Probes 

Single-stranded piece of DNA, tagged with an identifiable marker, that is used to detect a complementary sequence; locates nucleotide sequence characteristic of species or group; most methods first increase DNA in sample (inoculation on agar or in vitro DNA amplification)

Fluorescence in situ hybridization (FISH) probes

Technique used to detect a given nucleotide sequence within intact cells on a microscope slide; used to determine if the DNA and the unknown DNA strand are bound; amplification step not needed due to the numerous natural copies of rRNA in multiplying cells; various different probes that bind rRNA are specific for a signature sequence

Signature sequence 

Characteristic nucleotide sequences in certain ribosomal RNA genes, or their products, that can be used to classify or identify certain organisms.

Nucleic Acid Amplification Tests (NAATs)

In vitro methods that are used to increase the number of copies of specific DNA sequences; allows detection of small numbers of organisms, often from body fluids, soil, food, water; the detection of organisms that cannot be cultured; the polymerase chain reaction (PCR) is a common technique

rRNAs

The nucleotide sequence of ribosomal RNA molecules; can be used to identify and classify microbes since its sequences are relatively stable; sequencing is a method used to identify microorganisms that requires amplifying and then sequencing genes, but it can also be used for organisms not yet grown in culture; small subunit (SSU) RNAs most useful

16S rRNA gene

Most useful because of moderate size (approximately 1,500 nucleotides), sequence compared with extensive databases, and can identify organisms that cannot be grown in culture

rDNAs

The DNA gene that encodes the nucleotide sequence of the rRNA molecule; rDNAs that encode rRNAs used due to ease of DNA sequencing methods; culturing not necessary (the gene can be amplified directly from DNA in environmental samples); environmental samples may be amplified by PCR; May not resolve at species level because closely related prokaryotes can have identical SSU rDNA sequences; DNA hybridization a better tool in these cases

Characterizing Strains

Important for foodborne illnesses, forensic investigations of bioterrorism, biocrimes, and diagnosing certain diseases

Biochemical Typing 

A method to characterise strains; commonly used to identify bacteria and distinguish different strains; a group of strains that have a characteristic pattern: biovar or biotype; can trace Vibrio

Biovar

A group of strains that have a characteristic biochemical pattern different from other strains; also called a biotype

Biotype

A group of strains that have a characteristic biochemical pattern different from other strains; also called a biovar

cholerae El Tor

A biochemical variant of Vibrio cholerae that caused a worldwide cholera epidemic in 1961

Serological Typing 

A method to characterise strains; a group of strains that have a characteristic antigen called a serovar or serotype; proteins and carbohydrates that cary among strains can be used for differentiation; E. coli distinguished by antigenic type of flagella, capsules, lipopolysaccharide molecules; E. coli O157:H7 (O antigen is lipopolysaccharide; H antigen is flagella)

Serovar

A group of strains that have a characteristic antigenic structure that differs from other strains; also called a serotype

Phage Typing

A method to characterise strains; relies on differences in susceptibility to bacteriophages (phages); susceptibility pattern determined by adding different bacteriophage suspensions to agar plate growing bacteria, if phage lyses cells, a clear area forms; largely replaced by molecular methods, but still useful in labs lacking equipment for molecular typing

Whole Genome Squencing (WGS)

A method to characterise strains and identify microorganisms; advances in DNA sequencing methods have made it easier to detect subtle differences among phenotypically identical strains; this is useful for identifying an isolate when other methods have failed, and to study disease outbreaks; replaced restriction fragment polymorphisms (RFLPS)

Restriction fragment polymorphisms (RFLPS)

Compares patterns of fragment sizes produced when the same restriction enzyme is used to digest an isolate’s DNA

PulseNet

A CDC network that tracks foodborne disease outbreaks; laboratories from around the country can submit WGS data from foodborne pathogens to a computerized database

Genome Trakr

An FDA-associated network of laboratories; collects WGS data used to track outbreaks of foodborne pathogens; data from PulseNet are shared

Global Microbial Identifier

a database of WGS data from around the world

Antibiogram Typing

A method to characterise strains; reveal differences in susceptibility to antimicrobial medications; bacteria grown on agar plate; discs containing different antimicrobials placed on agar; if cells are susceptible, a clear area forms; largely replaced by molecular methods

Sequence Analysis of Ribosomal Components

Reliable indicators because ribosomes perform crucial and functionally constant tasks in all organisms; only a limited number of mutations can be tolerated; some sequences will be similar even if organisms diverged long ago; ribosomal genes are not commonly horizontally transferred

DNA-DNA Hybridization

Relatedness of organisms determined by similarity of nucleotide sequences; measured by how completely single strands of their DNA hybridize with each other; extent of DNA-DNA hybridization reflects degree of sequence similarity; bacterial strains with over 70% similarity are considered members of the same species (Shigella and Escherichia would be grouped in same species based only on DNA hybridization)

Sequence Analysis of Genomes

As WGS data accumulate it may prove to be a more valuable tool than DNA-DNA Hybridization; to assess the relatedness of two isolates, shared genes of genome sequences are compared, and a measure called the average nucleotide identity (ANI) is then calculated

G + C content

Percentage of G-C base pairs in DNA; if content differs by more than a few percent, organisms are not related; similarity does not guarantee relatedness; DNA with higher percentage melts at a higher temperature due to 3 hydrogen bonds between the bases