Laboratory Standards Institute (CLSI) for mycobacterial identification do not recommend the use biochemical testing and instead
rely on other technologies such as matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry (MALDI-TOF MS),
defined algorithms for mycobacterial identification, and support
the use of molecular sequencing methods. These methods provide
faster, reproducible, and more accurate results that allow clinicians
to respond to the increasing incidence of infections with nontuberculous mycobacteria [10].
Genomic analysis of mycobacterial species shows a distribution
into five distinct monophyletic groups, described as the “Tuberculosis-Simiae,” “Terrae,” “Triviale,” “Fortuitum-Vaccae,” and the
“Abscessus-Chelonae” clades. These clades are the basis of the
recent taxonomic reclassification of the genus into five genera:
Mycobacterium, Mycolicibacter, Mycolicibacillus, Mycolicibacterium,
and Mycobacteroides, respectively [12]. While some have argued
that these results do not justify the “disruption” caused by this
nomenclatural reclassification, the phylogeny of these genera/
clades is clear and supported by other studies [13–15] (see Subheading 4.4 for additional details). For the sake of simplicity in this
chapter, the term mycobacteria refers to these five genera.
In order to describe some of the challenges faced by mycobacteriology laboratories in identification of mycobacteria, a summary
of results obtained by BEI Resources when producing several
mycobacterial isolates is presented. As part of its standardized
quality control procedure, BEI Resources characterizes mycobacterial items according to both phenotypic assays (including cell and
colony morphology, Gram and acid-fast staining, motility, pigment
production, growth at different temperatures, number of days
required by the item to grow, urease and catalase activity, nitrate
production, etc.) and genotypic assays (e.g., 16S rRNA or hsp65
sequencing) to provide species identification. The species-level
identification of some recently deposited strains proved to be challenging, initially delaying the release of these items to the catalog
(BEIresources.org). The results of phenotypic testing are shown in
Table 1.
The results of genotypic testing using 16S rRNA sequencing
for these items are shown in Table 2. Conventional genetic identification is typically performed using 16S rRNA sequencing, and the
sequenced gene is then compared with a database containing 16S
sequences, e.g., GenBank or PATRIC. To be considered as a match
to species, the sequence identity of the test sample should be
!98.7% to the 16S sequence of a species type strain [16]. More
generally, an identity percentage at or above this threshold indicates
that both the query strain and the reference strain belong to the
same species. However, because the reference strain could be misclassified in the database, a hit to a reference sequence should not be
considered definitive unless it is derived from the type strain of
Mycobacterial ID and Characterization Using WGS
401
rely on other technologies such as matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry (MALDI-TOF MS),
defined algorithms for mycobacterial identification, and support
the use of molecular sequencing methods. These methods provide
faster, reproducible, and more accurate results that allow clinicians
to respond to the increasing incidence of infections with nontuberculous mycobacteria [10].
Genomic analysis of mycobacterial species shows a distribution
into five distinct monophyletic groups, described as the “Tuberculosis-Simiae,” “Terrae,” “Triviale,” “Fortuitum-Vaccae,” and the
“Abscessus-Chelonae” clades. These clades are the basis of the
recent taxonomic reclassification of the genus into five genera:
Mycobacterium, Mycolicibacter, Mycolicibacillus, Mycolicibacterium,
and Mycobacteroides, respectively [12]. While some have argued
that these results do not justify the “disruption” caused by this
nomenclatural reclassification, the phylogeny of these genera/
clades is clear and supported by other studies [13–15] (see Subheading 4.4 for additional details). For the sake of simplicity in this
chapter, the term mycobacteria refers to these five genera.
In order to describe some of the challenges faced by mycobacteriology laboratories in identification of mycobacteria, a summary
of results obtained by BEI Resources when producing several
mycobacterial isolates is presented. As part of its standardized
quality control procedure, BEI Resources characterizes mycobacterial items according to both phenotypic assays (including cell and
colony morphology, Gram and acid-fast staining, motility, pigment
production, growth at different temperatures, number of days
required by the item to grow, urease and catalase activity, nitrate
production, etc.) and genotypic assays (e.g., 16S rRNA or hsp65
sequencing) to provide species identification. The species-level
identification of some recently deposited strains proved to be challenging, initially delaying the release of these items to the catalog
(BEIresources.org). The results of phenotypic testing are shown in
Table 1.
The results of genotypic testing using 16S rRNA sequencing
for these items are shown in Table 2. Conventional genetic identification is typically performed using 16S rRNA sequencing, and the
sequenced gene is then compared with a database containing 16S
sequences, e.g., GenBank or PATRIC. To be considered as a match
to species, the sequence identity of the test sample should be
!98.7% to the 16S sequence of a species type strain [16]. More
generally, an identity percentage at or above this threshold indicates
that both the query strain and the reference strain belong to the
same species. However, because the reference strain could be misclassified in the database, a hit to a reference sequence should not be
considered definitive unless it is derived from the type strain of
Mycobacterial ID and Characterization Using WGS
401