Advanced Techniques in Diagnostic Microbiology

20
Bacterial Identification Based
on 16S Ribosomal RNA Gene
Sequence Analysis
XIANG Y. HAN
Introduction
Clinical microbiology laboratory is responsible for the isolation or detection of
microorganisms to establish the diagnosis of infection. Rapid and accurate identification
of these organisms and subsequent antibacterial drug susceptibility tests
also guide antibiotic therapy. Although these goals can be fulfilled most of the
time, some bacteria may be difficult to be identified due to fastidious growth, morphological
variations, unusual biochemical reactions, lack of previous recognition,
or a combination of these. Subculture failure, though it rarely happens, virtually
makes routine identification impossible. Fortunately, technological advances have
largely overcome these limitations for bacterial identification. One of the advances
realized in the past decade or so has been the analysis of the nucleotide sequences
of the 16S ribosomal RNA gene (16S rDNA), which has emerged as the single best
method to identify bacteria (Kolbert and Persing, 1999; Drancourt et al., 2000).
This chapter reviews its theoretical basis, methodology, clinical application, and
limitations. A thorough and in-depth review with many practical points has just
been published elsewhere (Clarridge, 2004).
Theoretical Basis
Ribosomes are protein synthesis machines for living organisms and are required for
survival. Many antibiotics target the bacterial ribosome to achieve a bacteriocidal
effect. A bacterial ribosome is composed of multiple ribosomal proteins and three
ribosomal RNAs (rRNA) (i.e., 23S rRNA, 16S rRNA, and 5S rRNA). The rRNAs
are encoded by their respective genes, usually organized as an operon, termed rrn,
in the genome. With the genomes of >100 various bacteria having been sequenced,
it is realized that a bacterial genome may have multiple rrn operons depending on
the size of the genome and the species. Table 20.1 lists several examples of the
number of rrn operons and genome size. Generally, every mega–base pair (Mbp)
contains 1–3 rrn (mean 1.93 and median 1.92).
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