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Chapter 27 : Sequence-Based Identification and Characterization of Mycobacteria

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Abstract:

Perhaps no other technique has revolutionized the identification and characterization of a single clinically relevant genus as much as the use of sequencing for mycobacterial diagnostics. This chapter provides a comprehensive account of the current use of DNA sequencing for the identification and characterization of mycobacteria in the clinical microbiology laboratory. The problems associated with phenotypic identification of mycobacteria were highlighted in 1996, when Springer and colleagues compared 34 isolates identified by both biochemical testing and 16S rRNA sequencing. There are no FDA-approved platforms at this time designed specifically for the sequence-based identification of microorganisms. However, there are a number of commercially available sequencing platforms, and laboratories are free to establish their own laboratory-developed (home brew) system for mycobacterial sequencing. Molecular determination of drug resistance by sequence analysis has been successfully used for and, in a limited way, for other mycobacteria. Drug resistance in is moderated by mutations in a handful of genes. Direct sequencing remains a challenge due to the paucity of mycobacteria in many specimen types especially in comparison with the amount of other bacteria present in complex matrices like sputum.

Citation: Wengenack N, Hall L. 2011. Sequence-Based Identification and Characterization of Mycobacteria, p 437-452. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch27
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FIGURE 1

Mycobacterial species identified in each decade from 1896 to 2008.

Citation: Wengenack N, Hall L. 2011. Sequence-Based Identification and Characterization of Mycobacteria, p 437-452. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch27
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Image of FIGURE 2
FIGURE 2

Workflow for mycobacterial sequencing.

Citation: Wengenack N, Hall L. 2011. Sequence-Based Identification and Characterization of Mycobacteria, p 437-452. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch27
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Image of FIGURE 3
FIGURE 3

Sample electropherogram of The target sequenced was the first 500-bp region of the 16S rRNA gene.

Citation: Wengenack N, Hall L. 2011. Sequence-Based Identification and Characterization of Mycobacteria, p 437-452. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch27
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Image of FIGURE 4
FIGURE 4

Schematic of the mycobacterial 16S-23S rRNA region. The 16S rRNA, 23S rRNA, and ITS region (16S-23S spacer) are depicted. The 16S rRNA target is 1,542 bp long and includes highly conserved regions for primer binding. The forward primer usually sits at base pair position 4 or 27, and the reverse primer sits near base pair 534. The hypervariable regions provide areas that permit species distinctions. Hypervariable region A corresponds to positions around 129 bp to 267 bp. Hypervariable region B corresponds to positions around 430 to 500 bp ( ).

Citation: Wengenack N, Hall L. 2011. Sequence-Based Identification and Characterization of Mycobacteria, p 437-452. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch27
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Image of FIGURE 5
FIGURE 5

Schematic of the pyrosequencing process. The reaction requires a single-stranded DNA template and a sequencing primer. Nucleotides are added to the reaction in a defined order, and the light generated by the enzymatic cascade resulting from pyrophosphate release following nucleotide incorporation is captured in the form of a pyrogram. The sequence produced is approximately 50 to 100 nucleotides in length. (Figure courtesy of Biotage Biosystems.)

Citation: Wengenack N, Hall L. 2011. Sequence-Based Identification and Characterization of Mycobacteria, p 437-452. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch27
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Tables

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TABLE 1

Targets used for sequencing of mycobacteria

Citation: Wengenack N, Hall L. 2011. Sequence-Based Identification and Characterization of Mycobacteria, p 437-452. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch27
Generic image for table
TABLE 2

species with indistinguishable 500-bp 16S sequences

Citation: Wengenack N, Hall L. 2011. Sequence-Based Identification and Characterization of Mycobacteria, p 437-452. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch27
Generic image for table
TABLE 3

Targets other than 16S used for sequencing of individual mycobacterial species

Citation: Wengenack N, Hall L. 2011. Sequence-Based Identification and Characterization of Mycobacteria, p 437-452. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch27
Generic image for table
TABLE 4

Gene loci conferring drug resistance in

Citation: Wengenack N, Hall L. 2011. Sequence-Based Identification and Characterization of Mycobacteria, p 437-452. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch27
Generic image for table
TABLE 5

Resistance markers for nontuberculous mycobacteria

Citation: Wengenack N, Hall L. 2011. Sequence-Based Identification and Characterization of Mycobacteria, p 437-452. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch27

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