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Chapter 29 : Laboratory Characteristics of Slowly Growing Mycobacteria

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Abstract:

This chapter represents that detailed phenotypic methods of identification of mycobacteria. Although some phenotypic characterization remains important, it is now well recognized by experts in mycobacterial taxonomy that current accurate identification of organisms of both the complex (MTBC) and the nontuberculous mycobacteria (NTM) requires molecular techniques for definitive identification to species level. The MTBC remains the most important group within the genus from a global and clinical perspective. species differ in the ability to grow at certain temperatures. For determination of the preferred growth temperature, solid culture media are inoculated with defined suspensions of mycobacteria and incubated at various temperatures. The chapter further highlights that some species cannot use nicotinic acid, although they produce it in their biosynthetic pathways. Commercially available assays are intended for the detection of some of the most important species and can be performed from both solid and liquid media. Two tests for the identification of several mycobacterial species are commercially available but are not yet FDA approved. The INNO-LiPA Mycobacteria v2 assay is based on the nucleotide differences in the 16S-23S rRNA gene spacer region. The GenoType Mycobacterium CM/AS test is based on the detection of species-specific sequences in the 23S rRNA gene. The major criterion for molecular strain typing is that the test isolates must all belong to the same species. Thus, definitive identification to the species level is an important prerequisite.

Citation: Richter E, Brown-Elliott B, Wallace R. 2011. Laboratory Characteristics of Slowly Growing Mycobacteria , p 503-524. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch29

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FIGURE 1

grown on a Löwenstein-Jensen slant for 14 days. Colonies are dry, wrinkled, off-white, and eugenic.

Citation: Richter E, Brown-Elliott B, Wallace R. 2011. Laboratory Characteristics of Slowly Growing Mycobacteria , p 503-524. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch29
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Image of FIGURE 2
FIGURE 2

on Löwenstein-Jensen slants after 2 weeks of growth before exposure to light (left) and after exposure to light (photochromogenic) (right).

Citation: Richter E, Brown-Elliott B, Wallace R. 2011. Laboratory Characteristics of Slowly Growing Mycobacteria , p 503-524. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch29
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Image of FIGURE 3
FIGURE 3

grown on 7H10 Middlebrook agar for 10 days. Colonies are yellow and smooth (scotochromogenic).

Citation: Richter E, Brown-Elliott B, Wallace R. 2011. Laboratory Characteristics of Slowly Growing Mycobacteria , p 503-524. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch29
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Image of FIGURE 4
FIGURE 4

grown on 7H10 Middlebrook agar for 10 days. Colonies are small, colorless, and smooth (nonchromogenic).

Citation: Richter E, Brown-Elliott B, Wallace R. 2011. Laboratory Characteristics of Slowly Growing Mycobacteria , p 503-524. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch29
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Image of FIGURE 5
FIGURE 5

Line probe assay (GenoType Mycobacterium CM/AS) for the identification of species. CC, conjugate control; UC, universal control; GC, genus-specific control; 4 to 17, specific bands for identification of the species; M, marker line (flags the upper side of the strip). Lines 1 to 6, CM strips; lines 7 and 8, AS strips. First lane, second lane, MTBC; third lane, fourth lane, fifth lane, sixth lane, negative control; seventh lane, eighth lane,

Citation: Richter E, Brown-Elliott B, Wallace R. 2011. Laboratory Characteristics of Slowly Growing Mycobacteria , p 503-524. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch29
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Tables

Generic image for table
TABLE 1

Characteristics of currently recognized species of NTM

Most strains (>99%) are nonpigmented.

Birds.

Sequence outside the 441-bp Telenti fragment.

Humans and swine.

—, not grown on artificial media.

Eels.

Fish.

Y, yes; N, no; U, undetermined; Q, questionable.

Citation: Richter E, Brown-Elliott B, Wallace R. 2011. Laboratory Characteristics of Slowly Growing Mycobacteria , p 503-524. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch29
Generic image for table
TABLE 2

Important properties of selected slowly growing NTM species

Clinical relevance in pulmonary specimens must be proven according to the ATS guidelines.

AccuProbe test is positive for MAC and for -specific probes but negative for -specific probe.

AccuProbe test is positive for MAC-specific probe but negative for - and for -specific probes.

No data available for AccuProbe tests.

Sequence data exists only for a fragment of the gene.

Different reports with inconsistent data.

See reference . The test method differed from the current strip method.

Abbreviations: Neg, negative; Pos, positive; ND, not determined; Var, varies.

Citation: Richter E, Brown-Elliott B, Wallace R. 2011. Laboratory Characteristics of Slowly Growing Mycobacteria , p 503-524. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch29
Generic image for table
TABLE 3

Commercially available NAA tests

Received FDA approval for smear-positive respiratory specimens and smear-negative respiratory specimens from patients suspected of having TB.

Received FDA approval for smear-positive respiratory specimens from patients suspected of having TB.

Citation: Richter E, Brown-Elliott B, Wallace R. 2011. Laboratory Characteristics of Slowly Growing Mycobacteria , p 503-524. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch29
Generic image for table
TABLE 4

Phenotypic identification of , and BCG

On Löwenstein-Jensen medium.

TCH, thiophen-2-carboxylic acid hydrazide (tested on solid Löwenstein-Jensen medium).

Preferrably tested with the BACTEC 460 or BACTEC MGIT 960 method.

Some strains are weakly positive.

Isoniazid-resistant strains are also resistant to TCH.

Can be resistant if the strain is already resistant to other first-line drugs.

Citation: Richter E, Brown-Elliott B, Wallace R. 2011. Laboratory Characteristics of Slowly Growing Mycobacteria , p 503-524. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch29
Generic image for table
TABLE 5

Primers for amplification and sequencing of the mycobacterial 16S rRNA gene

Position in the ribosomal 16S rRNA gene.

F, forward; R, reverse.

Citation: Richter E, Brown-Elliott B, Wallace R. 2011. Laboratory Characteristics of Slowly Growing Mycobacteria , p 503-524. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch29
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TABLE 6

Primers for amplification and sequencing of the mycobacterial ITS 1

Citation: Richter E, Brown-Elliott B, Wallace R. 2011. Laboratory Characteristics of Slowly Growing Mycobacteria , p 503-524. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch29
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TABLE 7

Primers for amplification of a fragment of the gene of MTBC

Citation: Richter E, Brown-Elliott B, Wallace R. 2011. Laboratory Characteristics of Slowly Growing Mycobacteria , p 503-524. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch29
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TABLE 8

Position of characteristic SNPs within the gene

The SNP is in boldface.

Citation: Richter E, Brown-Elliott B, Wallace R. 2011. Laboratory Characteristics of Slowly Growing Mycobacteria , p 503-524. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch29
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TABLE 9

MIRU-VNTR patterns of H37Rv and BCG (12-locus analysis)

According to Supply et al. ( ).

V, variable among different BCG strains.

Citation: Richter E, Brown-Elliott B, Wallace R. 2011. Laboratory Characteristics of Slowly Growing Mycobacteria , p 503-524. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch29

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