Medical Microbiology
 
Bacterial Classification

      Understanding the relevance and complex nomenclature of literally hundreds of "important" bacteria can be challenging. The mastery of this exercise depends on the systematic organization of the bewildering array of different organisms into logical relationships (i.e., the taxonomic classification of organisms).

Phenotypic Classification

      The microscopic and macroscopic morphologies of bacteria were the first characteristics used to identify bacteria and form the cornerstones for most identification algorithms used today. For example, bacteria can be classified by their ability to retain the Gram stain (Gram positive or Gram negative) and by the shape of the individual organisms (cocci, bacilli, curved, or spiral). The macroscopic appearance of colonies of bacteria can also be used to identifv bacteria (e.g., hemolytic properties on agar containing blood, pigmentation of the colonies, size and shape of the colonies). Thus, Streptococcus pyogenes is a Gram positive bacterium that forms long chains of cocci and appears as small, white, hemolytic colonies on blood agar plates. Because many organisms can appear very similar on microscopic and macroscopic examination, morphologic characteristics are used to provide a tentative identification of the organism and to select more discriminating classification methods.
      The most common methods that are still used to identify bacteria consist of measuring the presence or absence of specific biochemical markers (e.g., ability to ferment specific carbohydrates or use different compounds as a source of carbon for growth; presence of specific proteases, lipases, or nucleases; presence of various aminopeptidases). With the use of carefully selected biochemical tests, most clinically significant isolates can be identified with a high degree of precision. These methods have also been used for subdividing groups of organisms beyond the species level, primarily for epidemiologic purposes (e.g., to determine whether a group of organisms from the same genus and species is from a common source or from distinct sources). These techniques are referred to as biotyping.
      Many bacteria possess antigens that are unique, and antibodies used to detect these antigens are powerful tools for their identification (serotyping). These serologic tests can be used to identify organisms that are inert in biochemical testing (e.g., Francisella, the organism that causes tularemia), difficult or impossible to grow (e.g., Treponema pallidum, the organism responsible for syphilis), associated with specific disease syndromes (e.g., Escherichia coli serotype O157, responsible for hemorrhagic colitis), or need to be identified rapidly (e.g., S. pyogenes, responsible for streptococcal pharyngitis). Serotyping is also used to subdivide bacteria below the species level for epidemiologic purposes.
      Other examples of phenotypic methods used to classify bacteria include analysis of antibiogram patterns (patterns of susceptibility to different antibiotics) and phage typing (susceptibility to viruses that infect bacteria-bacteriophages). Assessment of antibiotic susceptibility patterns is commonly performed but has limited discriminatory power. Phage typing is technically cumbersome and has now been replaced by more sensitive genetic techniques.

Phenotypic Classification Methods
      - Microscopic morphology
      - Macroscopic morphology
      - Biotyping
      - Serotyping
      - Antibiogram patterns
      - Phage typing

Analytic Classification

      Analysis of the analytic characteristics of bacteria has also been used to classify bacteria at the genus, species, or subspecies level. The chromatographic pattern of cell wall mycolic acids is unique for many of the individual species of mycobacteria and has been used for more than 25 years to identify the most commonly isolated species. Analysis of the lipids in the entire cell has also proved to be a useful method for characterizing many bacterial species, as well as yeasts. Analyses of the whole cell proteins and cellular enzymes (multilocus enzyme electrophoresis) are also techniques that have been used to characterize bacteria, most typically at the subspecies level for epidemiologic investigations. Although these analytic methods are accurate and reproducible, they are labor intensive, and the instrumentation is expensive. For these reasons the analyses are used primarily in reference laboratories.

Analytic Classification Methods
      - Whole cell lipid analysis
      - Whole cell protein analysis
      - Multifocus locus enzyme electrophoresis
      - Cell wall fatty acid analysis

Genotypic Classification

       The most precise method for classifying bacteria is by analysis of their genetic material. Organisms were initially classified by the ratio of guanine to cytosine; this procedure has largely been forsaken for more discriminating methods, however. DNA hybridization was used initially to determine the relationship among bacterial isolates (e.g., to determine whether two isolates were in the same genus or species). More recently, this technique has been exploited for the rapid identification of organisms by use of molecular probes. That is, DNA from an organism to be identified is extracted and exposed to species-specific molecular probes. If the probe binds to the DNA, then the organism's identity is confirmed. This technique has also been used to detect organisms directly in clinical specimens, thus avoiding the need to grow the organisms. DNA hybridization has proved to be a valuable tool for the rapid detection and identification of slow growing organisms such as mycobacteria and fungi.
      An extension of the hybridization method is nucleic acid sequence analysis. Probes are used to localize specific nucleic acid sequences that are unique to a genus, species, or subspecies. These sequences are amplified so that millions of copies are produced, and then the amplified genetic material is sequenced to define the precise identity of the isolate. This method primarily analyzes sequences of ribosomal DNA (because highly conserved [family- or genus-specific] sequences and highly variable [species- or subspecies-specific] sequences are present). It has also been used to define the evolutionary relationship among organisms and to identify organisms that are difficult or impossible to grow. Most of the recent changes in taxonomic nomenclature were determined by nucleic acid sequence analysis. An extension of this work is the complete sequencing of a bacterium's entire genome, a technique that has now become technically feasible.
      Various other methods have been used, primarily to classify organisms at the subspecies level for epidemiologic investigations: plasmid analysis, ribotyping, and analysis of chromosomal DNA fragments. In recent years, the technical aspects of these methods have been simplified to the point that most clinical laboratories use variations of these methods in their day-to-day practice.

Genotypic Classification of Bacteria
      - Guanine and cytosine ratio
      - DNA hybridization
      - Nucleic acid sequence analysis
      - Plasmid analysis
      - Ribotyping
      - Chromosomal DNA fragment