Much like letters, words, sentences, and paragraphs in a book, the sequences of nucleic acids in DNA and RNA tell a story about the genetic capabilities of an isolated organ ism. At the highest level, a sequence of DNA or RNA can be selected that is used to identify an organism at the genus or species level or characterize a gene encoding a virulence marker or antibiotic resistance. At a deeper level, the sequences of DNA can be used to subtype an organism for epidemiologic purposes. In this section, only the most commonly used techniques for identification and epidemiologic subtyping organisms are discussed.
NUCLEIC ACID SEQUENCING
Sequencing can be subdivided into targeted sequencing, in which a specific region of DNA or RNA is sequenced, and whole genome sequencing (WGS) in which the entire microbial genome is sequenced. Targeted sequencing is used primarily to identify an organism or detect a virulence gene or antibiotic resistance gene in a sample, whereas WGS is used for subtyping or genotyping organisms. Sequencing the ribosomal RNA genes is a common procedure for the definitive identification of bacteria and fungi because portions of these genes are highly conserved and useful for microbial identification at the genus level, whereas other sequences of the ribosomal RNA genes are species specific. Likewise, comparison of the genome sequences of individual bacteria is a common method for assessing their degree of relatedness. With each replication, one or more mutations are potentially introduced in the genomes of the bacterial progeny. Thus the more mutations (single nucleotide polymorphisms [SNPs]) that are present in the genomes, the more distantly related are the two organisms. This procedure has become the gold standard for investigations of infectious outbreaks. However, even though the process of WGS, including sequencing long nucleic acid fragments and then assembly into complete genomes, has become relatively simple and inexpensive in recent years, most laboratories still do not have the technical expertise to use these tools. Thus a variety of other methods are used for epidemiologic classification, with the most commonly used method described below.
RESTRICTION FRAGMENT LENGTH POLYMORPHISM
Specific strains of microorganisms (primarily bacteria and viruses) can be distinguished based on the size of DNA fragments produced when the genomic DNA is cleaved by specific restriction endonucleases (restriction enzymes), enzymes that recognize and cleave the DNA at specific DNA sequences. The cleavage usually results in a characteristic number of DNA fragments of different lengths. The pat tern of DNA fragments may differ between strains (restriction fragment length polymorphism [RFLP]) and is used to determine the relatedness of different strains (Fig. 1). DNA fragments of different sizes or structures can be distinguished by their electrophoretic mobility in an agarose or polyacrylamide gel. The DNA fragment moves through the maze-like structure of the gel at different speeds, allowing their separation. The DNA can be visualized by fluorescent staining with ethidium bromide. Smaller fragments (< 20,000 base pairs), such as those from bacterial plasmids or viruses, can be separated and distinguished by normal electrophoretic methods. Larger fragments, such as those from whole bacteria, can be separated by using a special electrophoretic technique called pulsed-field gel electrophoresis. The procedure for this technique is like standard gel electrophoresis except the current that moves the DNA fragments through the gel is alternated periodically. By doing this, very large fragments of DNA can move through the gel and be separated by size. RFLP is a labor intensive procedure that does not have the resolving power of WGS or SNP analysis. It is likely that this procedure will be discontinued in the next decade as sequencing techniques become more widely adopted in clinical microbiology laboratories.
Fig1. Restriction fragment length polymorphism distinction of DNA from bacterial strains separated by pulsed-field gel electrophoresis. Lanes 1–3 show Sma 1 restriction endonuclease-digested DNA from bacteria from two family members with necrotizing fasciitis and from their physician (pharyngitis). Lanes 4–6 are from unrelated Streptococcus pyogenes strains. (Courtesy Dr. Joe DiPersio, Akron, Ohio.)
