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  • CLINICAL MICROBIOLOGY REVIEWS, Oct. 2004, p. 840–862 Vol. 17, No. 4 0893-8512/04/$08.00�0 DOI: 10.1128/CMR.17.4.840–862.2004

    Impact of 16S rRNA Gene Sequence Analysis for Identification of Bacteria on Clinical Microbiology and Infectious Diseases

    Jill E. Clarridge III* Department of Laboratory Medicine, University of Washington, and Pathology and Laboratory

    Medicine Service, Veterans Affairs Medical Center, Seattle, Washington

    INTRODUCTION .......................................................................................................................................................840 MECHANICS OF THE PROCESS ..........................................................................................................................841

    Choice of the 16S rRNA Gene as the Gene To Sequence .................................................................................841 Basics of Sequencing ..............................................................................................................................................844

    ASSIGNING AN IDENTIFICATION WITH 16S rRNA GENE SEQUENCES .................................................845 Overview of Bacterial Identification and Taxonomic Placement Using 16S rRNA Gene Sequences .........845 Problems in Generating a Sequence ....................................................................................................................845 Generating Dendrograms and Comparing Sequences.......................................................................................846 Sequence Databases................................................................................................................................................848

    THE CLINICAL MICROBIOLOGIST’S DILEMMA IN ASSIGNING A GENUS AND SPECIES NAME..849 Common Definitions of Genus or Species Derived by 16S rRNA Gene Sequence Analysis ........................849 Problems with the Present Nomenclature...........................................................................................................850 Microheterogeneity in the 16S rRNA Gene Sequence Is Common..................................................................852

    MAJOR IMPROVEMENTS IN CLINICAL MICROBIOLOGY PRACTICE BY IDENTIFYING BACTERIA BY SEQUENCE INSTEAD OF PHENOTYPE .........................................................................854

    16S rRNA Gene Sequences Can Better Identify Poorly Described, Rarely Isolated, or Phenotypically Aberrant Strains .................................................................................................................................................854

    16S rRNA Gene Sequences Can Be Routinely Used for Identification of Mycobacteria .............................856 16S rRNA Gene Sequence Analysis Can Lead to the Discovery and Description of Novel Pathogens......856 16S rRNA Gene Sequence Analysis Can Identify Noncultured Bacteria........................................................857

    COSTS IN A ROUTINE CLINICAL MICROBIOLOGY LABORATORY.........................................................857 STANDARDS FOR EDITORS, REVIEWERS, AND LABORATORIANS..........................................................858 CONCLUSIONS .........................................................................................................................................................859 ACKNOWLEDGMENTS ...........................................................................................................................................859 REFERENCES ............................................................................................................................................................859

    INTRODUCTION

    One area within the practice of clinical microbiology is the craft of putting scientific names to microbial isolates. This is usually done with the intent of giving insight into the etiolog- ical agent causing an infectious disease, including pathological associations and possible effective antimicrobial therapy. The historical method for performing this task is dependent on the comparison of an accurate morphologic and phenotypic de- scription of type strains or typical strains with the accurate morphologic and phenotypic description of the isolate to be identified. Microbiologists authoring standard references such as Bergey’s Manual of Systematic Bacteriology or the Manual of Clinical Microbiology or compiling results from well-character- ized strains such as those found at the Centers for Disease Control and Prevention or the American Type Culture Collec- tion (ATCC) would publish tables summarizing the character- istics of each species of bacteria (35, 54, 60). Clinical microbi- ologists would try to match the results for their unknown clinical strain with a group in these tables. Not infrequently,

    there would be no perfect match and a judgment would have to be made about the most probable identification. Although various schema and computer programs were devised to help in these judgements, identification could vary among labora- tories (96).

    In the 1980s, a new standard for identifying bacteria began to be developed. In the laboratories of Woese and others, it was shown that phylogenetic relationships of bacteria, and, indeed, all life-forms, could be determined by comparing a stable part of the genetic code (111, 113). Candidates for this genetic area in bacteria included the genes that code for the 5S, the 16S (also called the small subunit), and the 23S rRNA and the spaces between these genes. The part of the DNA now most commonly used for taxonomic purposes for bacteria is the 16S rRNA gene (7, 36, 44, 52, 64, 101). The 16S rRNA gene is also designated 16S rDNA, and the terms have been used interchangeably: current ASM policy is that “16S rRNA gene” be used. The 16S rRNA gene can be compared not only among all bacteria but also with the 16S rRNA gene of ar- cheobacteria and the 18S rRNA gene of eucaryotes. Figure 1 shows the relationship of major branches of life, the Archaea, Bacteria (procaryotes), and Eucarya, as well as the major branches within the procaryotes based on these gene se- quences (62, 64, 111, 113).

    The goal of this review is to describe not only the mechanism

    * Mailing address: Pathology and Laboratory Medicine Services (113), VA Medical Center, 1660 S. Columbian Way, Seattle, WA 98108. Phone: (206) 277-4514. Fax: (206) 764-2001. E-mail: jill .clarridge@med.va.gov.

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  • and limits of bacterial 16S rRNA gene sequence analysis but also the impact and potential contribution that the 16S rRNA gene sequence analysis can make to the understanding of clin- ical microbiology and infectious diseases. It is hoped that this will promote recognition that the correct identification or tax- onomic name assignment can make a difference in our under- standing of the pathogenic process and in clinical outcome. A further goal is to help the clinical microbiologist winnow the enormous amount of taxonomic information now being gen- erated in order to promote meaningful and scientifically accu- rate communications with clinical colleagues.

    MECHANICS OF THE PROCESS

    Choice of the 16S rRNA Gene as the Gene To Sequence

    In the 1960s, Dubnau et al. (28) noted conservation in the 16S rRNA gene sequence relationships in Bacillus spp. Wide- spread use of this gene sequence for bacterial identification

    and taxonomy followed a body of pioneering work by Woese, who defined important properties. Foremost is the fact that it seems to behave as a molecular chronometer, as pointed out in an excellent review article by Woese (113). The degree of conservation is assumed to result from the importance of the 16S rRNA as a critical component of cell function. This is in contrast to the genes needed to make enzymes. Mutations in these genes can usually be tolerated more frequently since they may affect structures not as unique and essential as rRNA (if a bacterium does not have the gene to make the enzymes needed to utilize lactose, it can use an alternative sugar or protein as an energy source). Thus, few other genes are as highly con- served as the 16S rRNA gene. Although the absolute rate of change in the 16S rRNA gene sequence is not known, it does mark evolutionary distance and relatedness of organisms (44, 49, 62, 100). Problems in assigning a numerical value to this rate of change include the possibility that this rate of change of

    FIG. 1. Universal phylogenetic tree based on the 16S rRNA gene sequence comparisons. Reprinted from reference 62 with permission of the publisher.

    VOL. 17, 2004 16S rRNA GENE SEQUENCE FOR BACTERIAL IDENTIFICATION 841

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  • 16S rRNA gene may not be identical for all organisms (differ- ent taxonomic groups could have different rates of change), the rates could vary at times during evolution, and the rates could be different at different sites throughout the 16S rRNA gene. There are so-called “hot spots” which show larger numbers of mutations (101, 104); these areas are not the same for all species. 16S rRNA is also the target for several antimicrobial agents. As such, mutations in the 16S rRNA gene can affect the susceptibility of the organism to these agents and the 16S rRNA gene sequence can distinguish phenotypic resistance to antimicrobial agents (69, 70). However, these characteristics do not obviate or affect the use of 16S rRNA gene sequence for bacterial identification or assignment of close relationships at the