A Systematic Approach for Discovering Novel, Clinically Relevant Bacteria

Robert Schlaberg¹

, Keith E. Simmon¹, and Mark A. Fisher

Author affiliations: University of Utah School of Medicine, Salt Lake City, Utah, USA (R. Schlaberg, M.A. Fisher); ARUP Laboratories, Salt Lake City (R. Schlaberg, K.E. Simmon, M.A. Fisher)

Abstract

Sequencing of the 16S rRNA gene (16S) is a reference method for bacterial identification. Its expanded use has led to increased recognition of novel bacterial species. In most clinical laboratories, novel species are infrequently encountered, and their pathogenic potential is often difficult to assess. We reviewed partial 16S sequences from >26,000 clinical isolates, analyzed during February 2006–June 2010, and identified 673 that have <99% sequence identity with valid reference sequences and are thus possibly novel species. Of these 673 isolates, 111 may represent novel genera (<95% identity). Isolates from 95 novel taxa were recovered from multiple patients, indicating possible clinical relevance. Most repeatedly encountered novel taxa belonged to the genera Nocardia (14 novel taxa, 42 isolates) and Actinomyces (12 novel taxa, 52 isolates). This systematic approach for recognition of novel species with potential diagnostic or therapeutic relevance provides a basis for epidemiologic surveys and improvement of sequence databases and may lead to identification of new clinical entities.

Broad-range PCR amplification and sequencing of the 16S rRNA gene (16S sequencing) is not only widely used as a taxonomic tool but is recognized as an effective reference method for bacterial identification. It has been used to identify novel and emerging pathogens (1–4) and to define complex microbial communities (5,6). The method has also revolutionized our understanding of microbial diversity (7–9). In clinical microbiology laboratories, 16S sequencing is useful for classifying microorganisms from pure culture (10,11). Molecular identification is especially valuable for bacteria that are slow growing, biochemically inert or variable, and fastidious, and it has also enhanced our understanding of previously unrecognized, often opportunistic pathogens (1,10,12).
Sequence-based identification relies on limited, yet phylogenetically informative, 16S sequence variation between related bacterial taxa. The entire 16S rRNA gene is ≈1,500 nt long (11); however, sequencing the 5′ third (partial 16S) generally provides sufficient taxonomic information while limiting costs (10). Partial 16S sequences are compared with reference libraries to determine the species with maximum similarity (10,11). The largest library is the nucleotide database hosted by the National Center for Biotechnology Information (NCBI) (13). Depending on their similarity to reference sequences, unknown isolates can be identified to different taxonomic levels by using interpretive guidelines published by the Clinical and Laboratory Standards Institute (CLSI) (14). For most taxa, sequence identity >99% with a valid reference sequence is required for species-level identification. Although this cutoff is widely used to identify isolates of the same species, a uniform cutoff for defining isolates as belonging to separate species is more controversial (1,10,15–17). Values of 99.5% to 97.0% have been proposed in the past (12,15,17–22), with more recent evidence and recommendations supporting values between 98.7% and 99.0% (10,17,23).
In our laboratory, as in many others, 16S sequencing is performed when morphologic and phenotypic identification is inconclusive or difficult or when it is specifically requested. By using CLSI guidelines and an NCBI nucleotide-based reference library (24), >90% of these isolates can be identified to the species level. However, clinical isolates belonging to as-yet-undescribed taxa are regularly encountered. Whether they represent emerging pathogens (1) or environmental contaminants is often difficult to determine in individual cases. Therefore, we conducted a systematic analysis of large numbers of unidentified strains to screen for novel taxa of potential clinical relevance. We reviewed partial 16S sequences from >26,000 clinical isolates to identify and characterize novel species with possible clinical significance. We identified 673 isolates that belong to as-yet-undescribed species, including 348 isolates of 95 novel taxa that were isolated from multiple patients. Repeated isolation of these undescribed organisms may indicate their clinical relevance and warrant their formal description as species.