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J Clin Microbiol. Sep 2005; 43(9): 4311–4315.
PMCID: PMC1234117

Sequence-Based Identification of New Bacteria: a Proposition for Creation of an Orphan Bacterium Repository

Widespread use of gene sequencing for the identification of bacteria recovered from environmental and clinical specimens has increased dramatically the number of candidate new bacterial species of clinical importance (4). Some of the genes are shared by a vast majority, if not all, of bacterial species. The ribosomal 16S rRNA and 23S rRNA genes, the 16S-23S rRNA gene internal transcribed sequences (ITS) (9), housekeeping genes such as the rpoB gene encoding the β-subunit of RNA polymerase, the groEL gene encoding the heat-shock protein, the gyrB gene encoding the β-subunit of DNA gyrase (28), and homologous recombination-encoding recA are among such genes (Table (Table1).1). This offers possibilities of using these genes as “universal” or “broad-spectrum” targets for the identification of various bacterial species. All bacterial species possess at least one copy of the 16S rRNA gene, which contains highly conserved as well as hypervariable nucleic acid sequences (30). Therefore, the PCR targeting conserved nucleic acid sequences of 16S rRNA gene can be used to document the presence of bacteria in the clinical specimens or samples obtained from environmental sources. By the same measure, the targeting of hypervariable loci in the 16S rRNA gene enables the identification of the species of bacteria. Unlike phenotypic assays, the results of the PCRs targeting, as mentioned above, various nucleic acid sequences of 16S rRNA are not susceptible to interobserver variations. Furthermore, such results are transmissible via electronic database (e.g., GenBank [http://www.ncbi.nlm.nih.gov/GenBank], RIDOM [http://www.ridom-rdna.de]) (11, 29). Now, commercial test systems are available for the identification of bacterial species based on the nucleic acid sequences of 16S rRNA (MicroSeq; Applied Biosystems Inc., Foster City, Calif.) (10).

TABLE 1.
Contribution of molecular identification to the description of new bacterial species: the Mycobacterium sp. paradigm, 2004

In our laboratory, 0.5 to 1% of all bacterial isolates cannot be identified by their phenotypic characteristics; therefore, molecular methods are used for their identification (7). It was noted that identification by phenotypic assays may not be possible for as many as 14% of all clinical isolates of mycobacteria (24).

While the usefulness of sequencing of the 16S rRNA gene for the identification of bacterial species may not be in question, there still remain some unresolved issues (4). These include precise definition of the indications of using molecular methods for the identification of bacterial species, a generally acceptable cutoff value of 16S rRNA gene sequence similarity for tentative delineation of new bacterial species, and the nature of additional phenotypic and genotypic criteria for such delineation. Also, the minimum number of isolates that would be required for the description of a new bacterial species is not known.

WHEN SHOULD MOLECULAR IDENTIFICATION OF BACTERIA BE PERFORMED?

Apart from their central role in the detection and initial identification of as-yet-uncultured organisms in clinical specimens (12), 16S rRNA gene sequences have successfully been used for the identification of slow-growing organisms (e.g., Mycobacterium species) (10). Furthermore, sequencing of 16S rRNA gene is useful for the identification of the microorganisms that stain poorly. It may prove invaluable in the identification of microorganisms which are poorly reactive biochemically and show aberrant biochemical characteristics (7). Sequencing of the 16S rRNA gene may also explain the discrepancy between phenotypic identification and the antibiotic susceptibility profile of a microorganism (4).

In our laboratory, we used 16S rRNA gene sequences for the baseline identification of intracellular bacteria grown in shell-vial assays, such as Tropheryma whipplei (16). In clinical practice, one should be alert about the possibility of dealing with a novel pathogen if on initial testing the organism shows unusual phenotypic characteristics or antibiotic susceptibility patterns or both. In this situation, an identification system based on sequencing the 16S rRNA gene of the isolate may prove invaluable in unveiling its true identity (7).

WHEN DOES 16S rRNA GENE EVIDENCE SUGGEST A NEW BACTERIAL SPECIES IDENTIFICATION?

The sequences of the 16S rRNA gene of a particular organism may indicate that the organism either is an unusual phenotype belonging to a known taxon or represents a hitherto-unknown taxon (Fig. (Fig.1).1). It is believed that a 16S rRNA gene sequence similarity in the range of 0.5 to 1% would be required for the definition of a species (4). This corresponds to threshold values of 1% and 1.5% used by others to define a genus and species, respectively.

FIG. 1.
Proposed guidelines for the sequence-based identification of new bacteria by use of the 16S rRNA gene sequence as a first-line molecular tool.

We have proposed that two bacterial isolates would belong to different species if the similarity in the 16S rRNA gene sequence between them were less than 97%. In contrast, two bacterial isolates do not necessarily to belong to the same species if the similarity in 16S rRNA gene sequence between them is <3% (7). This is well illustrated by the fact that 16S rRNA gene sequences could not differentiate reliably two closely related Mycobacterium species (10). In this case, the same bacterial isolate contains multiple copies of the 16S rRNA gene with different sequences; sequencing of the 16S rRNA gene alone would not suffice for the identification of species (19). More-reliable results are likely to be obtained if both 16S rRNA and rpoB genes are sequenced (6). The rpoB gene, which encodes the beta-subunit of bacterial RNA polymerase, has been used for species delineation and identification of numerous genera, including mycobacteria (1, 18).

These data suggest that an original 16S rRNA gene or rpoB gene sequence exhibiting <97% similarity with its closest relative would constitute the basis for the description of a new bacterial taxon. The entire sequence of the 16S rRNA (1,500 bp) or rpoB gene (4,000 to 4,500 bp) containing <1% undetermined positions would be required for the description of a new taxon (23).

WHAT ADDITIONAL DATA WOULD BE REQUIRED TO PROPOSE A NEW BACTERIAL TAXON?

The sequence of the 16S rRNA gene may not be sufficient for the description of a new bacterial species. The source of isolation and, for clinical samples, tissue sites from which the samples have been obtained should be noted, as well as immunosuppression or history of exposure. Some basic and distinctive phenotypic characters should be included in the description of new taxons. These include conditions for optimum growth, the Gram-staining value, colony morphology, motility, spore-forming capacity, and median measurements as obtained by electron microscopic examination. Biochemical profiles of the isolate, including the results of oxidase and catalase testing and the ability to metabolize major carbohydrates, should also be useful for initial description of a taxon. The antibiotic susceptibility profile of the isolate should also be noted.

IS THERE A MINIMUM NUMBER OF STRAINS THAT WOULD BE REQUIRED FOR THE DESCRIPTION OF AN EMERGING BACTERIAL SPECIES?

The mere deposition of a 16S rRNA gene sequence in GenBank or similar databases will not ensure the description of a new species. This is mainly due to the fact that GenBank or similar databases do not define the minimum amount of data that a depositor must provide before the data can be stored in the database. Furthermore, the depositor may require his data be kept confidential for a period up to 1 year or until formal publication of the data. This would restrict the timely dissemination of data for the description of an emerging bacterial species. One way of circumventing this restriction is to publish the description of a new bacterial species in a peer-reviewed international biomedical journal.

There is as yet no consensus on the minimum number of isolates that would be required for the description of a new bacterial species. Formerly, International Bacteriology code did impose restrictions on the minimum number of isolates that would be required for the description of a new bacterial species. However, it has been recommended that the description of a new environmental bacterial species should be based on at least five strains derived from different geographic locations and environments (3, 13). Phenotypic and genotypic characters of newly described bacterial species should be compared with those of reference neighboring species and genera in order to identify precise phenotypic and genotypic characters that will discriminate the newly described bacterial species. Some laboratories still rely on biochemical profiles for the description of a bacterial species. However, an unusual phenotypic pattern may be considered an additional feature for the gene sequence-based description of emerging taxon.

Comparison of unusual phenotypic patterns of bacterial species reported from different settings is difficult because of the absence of an electronic database or mathematical model for such comparison. Therefore, as indicated above, for such comparisons a clinical microbiologist has to rely on published reports. Our data suggest that bacterial species representing an emerging species are no more frequent than one in every 4 years. At this frequency, a clinical microbiologist would see a new bacterial species every 20 years if the description of a new bacterial species were to be based on the inclusion of at least five bacterial isolates.

Our survey of the 2004 issues of the International Journal of Systematic and Evolutionary Microbiology revealed that although the actual number of bacterial isolates used for the description of a new species ranged between 1 and 72, in the majority of cases such description had been based on profiling a single isolate (Fig. (Fig.2).2). This held true in particular for slow-growing or fastidious organisms (e.g., T. whipplei, rickettsiae, ehrlichiae).

FIG. 2.
New species reported in the 2004 issues of the International Journal of Systematic and Evolutionary Microbiology (y axis) are plotted with the number of isolates used in the description of the species (x axis). Gray bars, new species of clinical interest; ...

All bacterial isolates having unusual genotypes or phenotypes or both should be reported. It is also important to confirm independently the identity of a newly described bacterial species by two laboratories.

SHOULD A NAME BE PROPOSED FOR AN EMERGING BACTERIAL SPECIES?

Naming of an emerging bacterial species is desirable. The name of an emerging bacterial species will indicate its source, pathogenicity, contagiousness, and antibiotic susceptibility pattern. A proper name for a bacterial species will facilitate interdisciplinary communication. There is no consensus on whether to use an acronym or Latinized name to designate a new bacterial isolate until its provisional name is validated. For example, the bacillus causing Whipple's disease was named Tropheryma whippelii even before it had been cultured in vitro. This initial nomenclature was based only on the DNA sequence of the organism (20). Subsequently, the bacillus causing Whipple's disease was renamed Tropheryma whipplei to Latinize it after the organism was cultured in vitro (16).

We propose that in accordance with current international convention, the discoverer of a new bacterial species should have the privilege of naming the bacterial species he has discovered. The Latinized name of a bacterial species may indicate the circumstances under which it has been discovered or its geographic source. However, a bacterial species should not bear the name of the patient from whom it has been isolated.

We also propose that only the microorganisms that have already been cultured should be given names and should be deposited in two international bacterial inventories. This is possible even for strictly intracellular bacteria. The American Type Culture Collection, Manassas, VA (http://www.atcc.org), as well as national collections in European countries (http://www.eccosite.org), offers such a possibility. Fastidious organisms can also be deposited in specialized collection centers. Our laboratory at the World Health Organization collaborative center for rickettsioses in Marseille is one such center (16).

PROPOSALS FOR AN ORPHAN BACTERIUM REPOSITORY

In order to clinical microbiologists in describing a new bacterial species, we propose to create an Internet-based repository for orphan isolates alongside sequence databases and international collections of bacteria. Such an electronic repository would incorporate an identity card with specific mentions of the collection number of the isolate in at least two international bacterial inventories, the source of isolation, phenotypic characters, antibiotic susceptibility pattern, complete 16S rRNA sequence with GenBank accession number and percentage of sequence similarity with the five closest relatives, any other significant gene sequence with GenBank accession number, any relevant criterion for the accurate identification, and the names and addresses of at least two corresponding authors (Table (Table22).

TABLE 2.
Proposal of items required for deposit of a new bacterial species into Electronic Orphan Bacteria Repositorya

Also, such an electronic repository would automatically offer the first depositor of a new bacterial species the opportunity to propose a name under a “Candidatus” status pending publication of the results in a peer-reviewed journal. Entries into such a database would be filed by microbiologists interested in the field of bacterial taxonomy up to the report of three to five isolates of potential new species. At that point, the automatic regulation system of the orphan isolate repository would electronically question the successive discoverers in the order of deposition with respect to their intention to describe the new species on the basis of collected data in the database in any relevant peer-reviewed journal, subject to contractual delay. The advantages of such an electronic repository would include normalization of criteria for the description of new bacterial species, reasonable enforcement of a three- to five-isolate rule, a shortened delay for precise description of new bacterial species, and a competitive system for such description among leading international groups. We propose that such an electronic repository should be held under the auspices of the American Society for Microbiology, alongside the relevant databases and bacterial collections, and named the Electronic Orphan Bacterium Repository, or EOBR, as its abbreviated form.

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