What is the easiest way to determine the species of non-tuberculous mycobacteria?

Question

We have isolated a mycobacterium strain from patient sputum using an NTM Elite plate.

When inoculated into an MGIT tube, we observe fluorescence when observed under UV light, which means it is highly likely to be a mycobacterium.

We have assumed it is M. abscessus since M abscessus has been grown from the same patient in the past. But what would be the easiest way to verify that?

Would the answer be different if we needed to know the subspecies, like M abscessus bolletii?

We are a small lab with limited equipment, so if the best solution is to use a service provider, we would also be open to that.

Answer 1

From this paper [Ref. 1] as well as this one [Ref. 2], it looks like PCR, potentially combined with genetic sequencing, is a good way to go.

In the first paper, they used 16S rRNA sequences to identify samples of non-tuberculous mycobacteria (NTM), and PCR restriction fragment length polymorphism analysis (PRA) of hsp65 to determine species. This method has the advantage of running on a standard PCR setup, then sequencing products of interest as needed (either in-house or via a service).

In the second paper they used a different method - multiplex real-time PCR with high-resolution melting (HRM). This method was performed on a RotorGene 600S instrument. It's not clear whether this particular instrument is necessary, or if the method can be adapted to any real-time PCR machine. This group also sequenced some samples.

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References:

1. Moghim S et al. Identification of Nontuberculous Mycobacteria Species Isolated from Water Samples Iran J Basic Med Sci. 2012 Sep-Oct; 15(5): 1076–1082. PMID: 23493797; PMCID: PMC3586926
2. Peixoto ADS et al. Identification of nontuberculous mycobacteria species by multiplex real-time PCR with high-resolution melting. Rev Soc Bras Med Trop. 2020 Nov 6;53:e20200211. doi: 10.1590/0037-8682-0211-2020. PMID: 33174954; PMCID: PMC7670742.