Concurrent Cultivation of Mycobacterium avium and Mycobacterium intracellulare Identified by a Single Sanger Sequencing of the 16S Gene

LETTER

Mycobacterium avium and Mycobacterium intracellulare are ubiquitous environmental mycobacteria that are frequently isolated from clinical specimens. These mycobacteria may cause significant infections, in immunocompromised patients in particular. Due to similar biochemical characteristics, they are usually grouped to and identified as Mycobacterium avium-intracellulare (MAI) complex without further differentiation. In studies and practices with separation of the two species, however, MAI differ considerably in natural niches and clinical significance (1–5).

At the 16S rRNA gene level, MAI differ by 11 nucleotides (0.7%) of the 1,530-bp gene. We used a PCR method to amplify and sequence a portion of the 16S gene to routinely identify various mycobacteria that were isolated in a cancer patient population (6). The method targeted the first 650 bp of the gene, the most variable regions known for mycobacteria. The primers were M13-AF1, 5′-TGTAAAACGACGGCCAGTGCTTAACACATGCAAGTCGA, and M13-AF3, 5′-CAGGAAACAGCTATGACCTGCAGTACTCTAGTCTGCCC, which were modified from the initial design by incorporation of the M13 primers (underlined) for sequencing purposes as well as redesign of the reverse primer AF-3 for robustness. The PCR sequencing method has been working well for positive cultures from solid media, such as colonies on LJ tubes and Middlebrook plates, as well as liquid media, such as pelleted bacilli from BacT/Alert MB bottles and Bactec blood culture bottles (the latter for rapidly growing mycobacteria). Of the 602-bp amplicon, MAI differ by 8 nucleotides, which enables confident separation. Here, we describe two instances in which MAI were cultivated simultaneously in the liquid medium and identified by one Sanger sequencing of the PCR amplicon.

The specimens included pleural fluid from a patient who had had esophageal surgery with subsequent anastomosis leak and a tracheal aspirate from a patient in intensive care. The specimens showed growth of mycobacteria in the BacT/Alert MB bottle upon incubation of 15 days and 9 days, respectively. The acid-fast bacilli in the liquid medium were pelleted by centrifugation, the DNA were extracted and amplified, and the amplicons were sequenced to resolve all intended 602 nucleotides with the exception of 8 ambiguities. BLAST analyses showed best and identical matches with both M. avium and M. intracellulare with mismatches of only the ambiguous nucleotides. Inspection of the electropherograms from both sequencing directions revealed dual peaks at the sites of ambiguity, which corresponded to the mismatched nucleotides between M. avium and M. intracellulare, suggesting the presence of both species. The sites and nucleotide peaks are shown in Fig. 1A. The liquid cultures were subcultured onto solid medium, resulting in typical colonies of the species—small clear entire M. avium and large raised pasty M. intracellulare (Fig. 1B)—as verified by separate sequencing reactions as well as seen consistently in our cultures.

FIG 1.

(A) Positions and Sanger sequencing peaks of the mismatched nucleotides in the first 300 bp of the 16S rRNA genes of M. avium and M. intracellulare. (B) Subculture showing the small clear colonies of M. avium and the large pasty colonies of M. intracellulare.

These findings allow us to recognize and report both M. avium and M. intracellulare in a timely fashion without subculture or additional sequencing. They also suggest similar density and growth rates of both species in the culture. Additionally, in institutions where the initial identification of MAI is rendered through the use of hybridization probes, the difference in colony morphology can be used to differentiate the species upon subculture.