Dichotomous Keys

Dichotomous keys organize bacterial traits in a manner that permits logical identification of organisms. The ideal system should contain the minimum number of features required for a correct categorization. Groups are split into smaller subgroups based on the presence (+) or absence (−) of a diagnostic character. Continuation of the process with different characteristics guides the investigator to the smallest defined subgroup containing the analyzed organism. In the early stages of this process, organisms may be assigned to subgroups based on characteristics that do not reflect genetic relatedness. It would be perfectly reasonable, for example, for a bacterial key to include a group such as “bacteria forming red pigments when propagated on a defined medium” even though this would include such unrelated forms as Serratia marcescens and purple photosynthetic bacteria. These two disparate bacterial assemblages occupy distinct niches and depend on entirely different forms of energy metabolism. Nevertheless, preliminary grouping of the assemblages would be useful because it would immediately make it possible for an investigator having to identify a red-pigmented culture to narrow the range of possibilities to relatively few groups. An example of a dichotomous key is shown in Figure 1.

Fig1. Algorithm for differentiating the Gram-positive cocci.

Numerical Taxonomy Using Biochemical Measures of Activity

Numerical taxonomy became widely used in the 1970s. These classification schemes use a number of unweighted, taxonomically useful characteristics. For these assays, an individual bacterial colony must be isolated and used to inoculate the test format. One example of this is the Analytical Pro file Index (API), which uses numerical taxonomy to identify a wide range of medically important microorganisms. APIs consist of several plastic strips, each of which has about 20 miniature compartments containing biochemical reagents (Figure 2). Almost all cultivatable bacterial groups and more than 550 different species can be identified using the results of these API tests. These identification systems have extensive databases of microbial biochemical reactions. The numerical clusters derived from these tests identify different strains at selected levels of overall similarity (usually >80% at the species level) based on the frequency with which they share traits. In addition, numerical classification provides percentage frequencies of positive character states for all strains within each cluster. The limitation of this approach is that it is a static system. As such, it does not allow for the evolution of bacteria and routine discovery of new bacterial pathogens.

Fig2. API test demonstrating how bacteria can be differentiated using a series of biochemical tests. Each small compartment contains a dehydrated powder that is inoculated from a bacterial culture. After incubation, the colorimetric changes can be scored numerically to produce a number that matches to a specific bacterial species and genus. (Courtesy of bioMerieux, Inc.)

Nucleic Acid–Based Taxonomy

 Since 1975, developments in nucleic acid isolation, amplification, and sequencing have spurred the evolution of nucleic acid–based subtyping systems. These include plasmid profile analysis, restriction fragment endonuclease analysis, repetitive sequence analysis, ribotyping, 16S ribosomal sequencing, and genomic sequencing. These methods are individually described as follows.

Plasmid Analysis

Plasmids are extrachromosomal genetic elements . These can be isolated from an isolated bacterium and separated by agarose gel electrophoresis to determine their number and size. Plasmid analysis has been shown to be most useful for examining outbreaks that are restricted in time and place (eg, an outbreak in a hospital), particularly when they are combined with other identification methods.

 Restriction Endonuclease Analysis

 The use of restriction enzymes to cleave DNA into discrete fragments is one of the most basic procedures in molecular biology. Restriction endonucleases recognize short DNA sequences (restriction sequences), and they cleave double stranded DNA within or adjacent to this sequence. Restriction sequences range from 4 to more than 12 bases in length and occur throughout the bacterial chromosome. Restriction enzymes that recognize short sequences (eg, 4-base pairs) occur more frequently than those that are specific for longer sequences (eg, 12-base pairs). Thus, enzymes that recognize short DNA sequences produce more fragments than enzymes that recognize infrequently occurring long DNA sequences. Several subtyping methods use restriction endonuclease digested DNA approaches.

One method involves isolating the plasmid DNA, which is generally of the size of several kilobases, and digesting this nucleic acid with a restriction enzyme. After enzymatic cleavage, fragmented plasmid segments are separated using agarose gel electrophoresis. Because plasmids carry genetic material that directly contribute to disease and are commonly moved from one organism to another, the presence of a common fragment may confirm that a specific bacterial isolate was identical to other isolates associated with an outbreak.

Another method involves the analysis of genomic DNA, which is of the size of several megabases. In this case, restriction endonucleases that cut at infrequently occur ring restriction sites within the bacterial genome are used. Digestion of DNA with these enzymes generally results in 5–20 fragments ranging from approximately 10 to 800 kb in length. Separation of these large DNA fragments is accomplished by a technique called pulsed field gel electrophoresis (PFGE), which requires specialized equipment. Theoretically, all bacterial isolates can be typed by this method. Its advantage is that the restriction profile consists of a finite number of well-resolved bands representing the entire bacterial chromosome in a single DNA fragment pattern.

Genomic Analysis

 In biology, the classic definition of a species is typically defined as the largest group of organisms in which two individuals can produce fertile offspring, typically by sexual reproduction. The multiplication of bacteria is almost entirely vegetative, and their mechanisms of genetic exchange rarely involve recombination among large portions of their genomes . Therefore, the concept of a species—the fundamental unit of eukaryotic phylogeny—has an entirely different meaning when applied to bacteria, making bacterial genome sequencing extremely useful.

The routine use of DNA genome sequencing allows the precise comparison of divergent DNA sequences, which can give a measure of their relatedness. Genes for different functions, such as those encoding surface antigens to escape immune surveillance, diverge at different rates relative to “housekeeping” genes such as those that encode cytochromes. Thus, DNA sequence differences among rapidly diverging genes can be used to ascertain the genetic distance between closely related groups of bacteria. Sequence differences among housekeeping genes then represent the related ness of widely divergent bacterial groups.

There is considerable genetic diversity among bacterial species. Chemical characterization of bacterial genomic DNA reveals a wide range of nucleotide base compositions among different bacterial species. One measure of this is the guanine + cytosine (G + C) content. If the G + C content of two different bacterial species is similar, it indicates taxonomic relatedness.

Repetitive Sequence Analysis

 In the current genomic era of molecular medicine, hundreds of microbial genomes have now been sequenced. With this era have come bioinformatical tools to mine this wealth of DNA sequence information to identify novel targets for pathogen subtyping, such as the repetitive sequences that have been found in different species . These repetitive sequences have been termed satellite DNA and have repeating units that range from 10 to 100 bp. They are commonly referred to as variable number tandem repeats (VNTRs). VNTRs have been found in regions controlling gene expression and within open reading frames. The repeat unit and the number of copies repeated side by side define each VNTR locus. A genotyping approach using polymerase chain reaction (PCR), referred to as multiple-locus VNTR analysis (MLVA), takes advantage of the levels of diversity generated by both repeat unit size variation and copy number among several well-characterized loci. It has proved especially useful in subtyping monomorphic species such as Bacillus anthracis, Yersinia pestis, and Francisella tularensis.

Ribosomal RNA sequencing

 Ribosomes have an essential role in protein synthesis for all organisms and as such are indisposable. Genetic sequence encodings both ribosomal RNAs (rRNA) and ribosomal proteins (both of which are required to comprise a functional ribosome) are highly conserved throughout evolution and have diverged more slowly than other chromosomal genes. Comparison of the nucleotide sequence of 16S rRNA from a range of prokaryotic sources revealed evolutionary relationships among widely divergent organisms and has led to the elucidation of a new kingdom, the archaebacteria. The phylogenetic tree based on rRNA data, showing the separation of bacteria, archaea, and eukaryote families, is depicted in Figure 3, which shows the three major domains of biological life as they are currently understood. From this diagram, two kingdoms, the eubacteria (true bacteria) and the archae bacteria, are distinct from the Eukaryotic branch.

Fig3. A phylogenetic tree based on rRNA sequencing data, showing the separation of bacteria, archaea, and eukaryotes families. The groups of the major known pathogenic bacteria are designated in gray. The only group of pathogenic bacteria that does not cluster in this shaded area is the Bacteroides group.

Ribotyping

 The technique of Southern blot analysis was named after its inventor, Edwin Mellor Southern, and has been used as a sub typing method to identify isolates associated with outbreaks.

For this analysis, DNA preparations from bacterial isolates are subjected to restriction endonuclease digestion. After aga rose gel electrophoresis, the separated restriction fragments are transferred to a nitrocellulose or nylon membrane. These double-stranded DNA fragments are first converted into single-stranded linear sequences. Using a labeled fragment of DNA as a probe, it is possible to identify the restriction fragments containing sequences (loci) that are homologous to the probe by complementation to the bound single-stranded fragments (Figure 4).

Fig4. Southern blot procedure showing how specific loci on separated DNA fragments can be detected with a labeled DNA probe. This procedure in essence allows for the discrimination of DNA at three levels: (1) at the level of restriction enzyme recognition, (2) by the size of the DNA fragment, and (3) by the hybridization of a DNA probe to a specific locus defined by a specific band at a specific position of the membrane.

Southern blot analysis can be used to detect polymorphisms of rRNA genes, which are present in all bacteria. Because ribosomal sequences are highly conserved, they can be detected with a common probe prepared from the 16S and 23S rRNA of a eubacterium (see Figure 5). Many organisms have multiple copies (five to seven) of these genes, resulting in patterns with enough fragments to provide good discriminatory power; however, ribotyping are of limited value for some microorganisms, such as mycobacteria, which have only a single copy of these genes.

Fig5. Electron micrograph of cells of a member of the mycoplasma group, the agent of bronchopneumonia in the rat (1960×). (Reproduced with permission from Klieneberger-Nobel E, Cuckow FW: A study of organisms of the pleuropneumonia group by electron microscopy. J Gen Microbiol 1955;12:99.)

مواضيع ذات صلة

Description Of The Major Categories And Groups Of Bacteria

الخصائص التصنيفية للبكتيريا

مقدمة عن تصنيف الاحياء المجهرية وعلاقاتها مع بعضها

المجاميع الانتقالية لوحدات التصنيف للاحياء المجهرية

التداخل بين الاحياء المجهرية

الأسس الجزئية المستعملة في تصنيف الاحياء المجهرية

التصنيف المقترح للأحياء المجهرية