Mycobacterium talmoniae, a Potential Pulmonary Pathogen Isolated from Multiple Patients with Bronchiectasis in the United States, Including the First Case of Clinical Disease in a Patient with Cystic Fibrosis

We characterize three respiratory isolates of the recently described species Mycobacterium talmoniae recovered in Texas, Louisiana, and Massachusetts, including the first case of disease in a patient with underlying cystic fibrosis. The three isolates had a 100% match to M. talmoniae NE-TNMC-100812^T by complete 16S rRNA, rpoB region V, and hsp65 gene sequencing.

ABSTRACT

We characterize three respiratory isolates of the recently described species Mycobacterium talmoniae recovered in Texas, Louisiana, and Massachusetts, including the first case of disease in a patient with underlying cystic fibrosis. The three isolates had a 100% match to M. talmoniae NE-TNMC-100812^T by complete 16S rRNA, rpoB region V, and hsp65 gene sequencing. Core genomic comparisons between one isolate and the type strain revealed an average nucleotide identity of 99.8%. The isolates were susceptible to clarithromycin, amikacin, and rifabutin, while resistance was observed for tetracyclines, ciprofloxacin, and linezolid. M. talmoniae should be added to the list of potential pulmonary pathogens, including in the setting of cystic fibrosis.

INTRODUCTION

Nontuberculous mycobacteria (NTM) are opportunistic pathogens that afflict both humans and animals and are often isolated from soil and water (1–4). The advancement of molecular methods, like multilocus sequence typing (MLST) and whole-genome sequencing (WGS), have helped in identifying more than 170 species of Mycobacterium, many of which cannot be definitively identified solely by biochemical or serological methods (5). Accurate species identification is important for the description of potential strict and opportunistic human pathogens and for determination of the treatment options by correlating the antimicrobial susceptibility testing profiles to the species/subspecies (6).

Mycobacterium talmoniae, recently described in 2017 (7) in Nebraska, is a slowly growing mycobacterium isolated from a human respiratory sample of a patient with no underlying disease. We have since recovered this species from two patients suffering from chronic bronchiectasis and one patient with cystic fibrosis and bronchiectasis over several years, the latter of which was the only confirmed clinical case. We report our findings of this organism along with its antimicrobial susceptibility testing results here for consideration of this being an emerging pathogen. We also report the first case of M. talmoniae clinical disease in a patient with cystic fibrosis (CF).

MATERIALS AND METHODS

Clinical histories.

In 2008, we recovered multiple sputum isolates of an undescribed slowly growing Mycobacterium (SGM) species from a patient with CF in Texas. We subsequently recovered similar sputum isolates from two other adult patients in 2015 from Louisiana and in 2016 from Massachusetts (see Table 1). The isolates of the first patient were initially identified by the hsp65-PCR restriction analysis (PRA) method as an unidentified nonpigmented SGM. Subsequently, MLST using partial 16S rRNA, rpoB region V, and hsp65 gene sequencing of all isolates from the three patients identified the isolates as the novel Mycobacterium species Mycobacterium talmoniae (7). Here, we describe clinical and molecular features of these isolates, including a case report of the first patient with confirmed clinical disease and outcome of drug therapy.

TABLE 1.

History of sputum isolates of Mycobacterium talmoniae^a

Serial no.	Strain	Yr	State	No. of isolates	Clinical disease	Underlying disease(s)
1	ATCC BAA-1052b	2000	Oregon	1	NA	NA
2	ATCC BAA-2683Tb	2012	Nebraska	Multiple	No	Chronic lung disease
3	Case 1	2008	Texas	6	Yes	CF, bronchiectasis
4	Case 2	2015	Louisiana	1	No	Bronchiectasis
5	Case 3	2016	Massachusetts	1	No	Bronchiectasis

^aNA, not available.

^bFrom the study by Davidson et al. (7).

The first case was a 10-year-old female in Texas with CF suffering from an unknown chronic mycobacterial nodular bronchiectatic lung disease. She had symptoms of increased cough and mucus production, and a high-resolution chest computed tomography (CT) scan was suggestive of NTM disease. She had six consecutive acid-fast bacilli (AFB) smear-positive sputum samples that were subsequently culture positive on solid and liquid media in an average of 10.8 days. Based on susceptibility testing results in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines (8), the patient was started on a daily three-drug regimen that consisted of clarithromycin at 250 mg twice daily, rifampin at 150 mg twice daily, and ethambutol at 400 mg twice daily. The MICs for this isolate are shown in Table 2. After approximately one month of treatment, her sputum was AFB smear negative and culture negative on both solid and broth media, and the patient showed clinical improvement, with diminished cough and mucus. The patient remained culture negative on therapy for 12 months, at which time her therapy was discontinued. The patient has now remained culture negative for this organism for more than 9 years.

TABLE 2.

Antimicrobial susceptibility of Mycobacterium talmoniae using broth microdilution MIC values of each isolate

	MIC (µg/ml)a
Antimicrobial	Case I	Case II	Case III	Type strain ATCC BAA-2683 (7)
Amikacin	4 (S)	2 (S)	2 (S)	2 (S)
Ciprofloxacin	4 (R)	4 (R)	8 (R)	4 (R)
Clarithromycin	<0.5 (S)	0.5 (S)	0.5 (S)	0.25 (S)
Doxycycline	ND	>16 (R)	>16 (R)	ND
Ethambutol	4 (I)	8 (R)	8 (R)	ND (I)
Linezolid	>64 (R)	>64 (R)	>64 (R)	>64 (R)
Minocycline	>32 (R)	ND	ND	ND
Moxifloxacin	2 (I)	1 (S)	2 (I)	2 (I)
Rifabutin	<0.006 (S)	<0.25 (S)	<0.25 (S)	0.25 (S)
Rifampin	1 (S)	1 (S)	1 (S)	2 (R)
Trimethoprim-sulfamethoxazole	1/19 (S)	1/19 (S)	2/38 (S)	ND

^aS, susceptible; R, resistant; I, intermediate; ND, not determined. MIC interpretations follow the Clinical and Laboratory Standards Institute (CLSI) document M24-A2 guidelines for antimicrobial susceptibility of nontuberculous mycobacteria (8).

The second case was a 73-year-old male referred for an evaluation of previously diagnosed Mycobacterium avium and Mycobacterium abscessus lung disease with chronic nodular bronchiectasis on high-resolution chest CT. The sputum was AFB smear negative and became culture positive for M. talmoniae in liquid medium only after 5 days. Five subsequent AFB cultures failed to grow the organism, the organism was considered not clinically significant, and the patient was not treated.

The third case was a 76-year-old female from Boston, MA, diagnosed with bronchiectasis and previously treated for Mycobacterium kansasii lung disease in 2012. Two years later, another SGM species, Mycobacterium lentiflavum, was recovered from an induced sputum sample. In 2016, an unidentified SGM subsequently identified as M. talmoniae was recovered from another induced sputum sample. The sample was AFB smear negative and grew in broth and solid media in 7 days. No additional cultures grew the SGM. The isolate was considered not clinically significant, and the patient was not treated.

Phylogenetic analysis.

Multilocus sequence typing (MLST) was performed on all the isolates from the three patients using full 16S rRNA, rpoB region V, and hsp65 gene sequencing, as previously described (9–12), and the results were analyzed using cutoff values suggested in references 9 and 10 and/or according to CLSI guidelines (13).

Sequences obtained from MLST of patient isolates from the three cases, the type strain of M. talmoniae, and select rapidly growing mycobacteria (RGM) and other SGM species were aligned, and phylogenetic trees were constructed in MEGA7 (14) using the neighbor-joining method and number of differences method (15); these are expressed in the units of the number of base differences per sequence.

Whole-genome sequencing.

Whole-genome sequencing was performed on an isolate from case 1. Sequencing libraries were prepared using 1 ng genomic DNA using Nextera XT with 14 cycles of dual-index PCR and then sequenced using a 2 × 260-bp run on an Illumina MiSeq platform (16, 17). Raw reads were quality and adapter trimmed using cutadapt, repaired using pairfq, de novo assembled using SPAdes version 3.8, and annotated using Prokka version 1.11 (18, 19). After trimming, a total of 180,533 paired-end reads were assembled into 381 scaffolds totaling 5,622,126 bp, with an N₅₀ value of 24,990 bp (GenBank accession number MLQM00000000).

Susceptibility testing.

Susceptibility testing of the patient isolates was performed using broth microdilution according to CLSI guidelines (8). The rifampin-resistant M. kansasii and other NTM (excluding M. avium complex) breakpoints were used for the susceptibility test interpretations.

Data availability.

For 16S rRNA, the GenBank nucleotide accession numbers are MH057081 to MH057088; for rpoB region V, the GenBank accession numbers are MH119336 to MH119343; and for hsp 65, the GenBank accession numbers are MH127921 to MH127928.

RESULTS

The gene sequences of the eight isolates from the three patients were 100% identical to each other by all three genes. By full 16S rRNA gene sequencing, all eight isolates were a 100% match to the type strain of M. talmoniae, ATCC BAA-2683 (DSM 46873; GenBank accession number KX008970). These also matched M. talmoniae ATCC BAA-1052 (GenBank accession number MG827408), a strain previously known as “Mycobacterium coloregonium” (7), Mycobacterium eburneum DSM 44358^T (20) (a novel species described after M. talmoniae) (GenBank accession number MG827408), and an unnamed Mycobacterium sp. from Germany submitted more than 20 years ago (GenBank accession number U65104).

Sequencing of the 441-bp Telenti fragment of the hsp65 gene and the region V rpoB gene yielded a 99.8% and 100% match to the M. talmoniae type strain, respectively. Phylogenetic trees were constructed using the neighbor-joining method and number of differences method (15) and are expressed in the units of the number of base differences per sequence.

The currently recognized closest related species to M. talmoniae include several taxa of RGM, such as the Mycobacterium fortuitum group, Mycobacterium mucogenicum, as well as SGM, e.g., Mycobacterium simiae, by full 16S rRNA gene analysis (see Fig. 1). M. talmoniae is located on a truly distinct branch of the 16S rRNA phylogenetic tree, clearly separated from all other SGM (7) and RGM, challenging a phylogenetic assignment to either group. Similarly, closest relatives by rpoB and hsp65 included several species of both SGM and RGM (Fig. 2 and 3), with M. talmoniae situated in an intermediary position. The finding of a match by 16S rRNA gene with another recently described species, M. eburneum, suggests the possibility that these represent the same species. M. eburneum, described as an RGM, was published only 1 month following that of M. talmoniae (20). By MLST, hsp65 gene sequences of the type strains of M. eburneum and M. talmoniae were a 100% match to each other, with few ambiguous bases noticed in our in-house-sequenced M. eburneum type strain from DSMZ. Also, by rpoB region III and V gene sequencing, the M. eburneum DSM 44358^T sequence did not match to that of the rpoB gene sequence of the M. eburneum type strain in GenBank (accession number KY630442). However, the rpoB region V sequence of the M. eburneum strain obtained from DSMZ, i.e., DSM 44358^T, is a 100% match to that of M. talmoniae NE-TNMC-100812^T, with two ambiguous bases in the M. eburneum rpoB gene sequence. DSMZ was notified of this discrepancy. By rpoB region III analysis, the M. eburneum DSM 44358^T strain has a 1-bp mismatch with both the M. eburneum and M. talmoniae type strains in GenBank. This may raise doubts that M. eburneum and M. talmoniae may be same species. Further analyses and conversations are required to resolve these inconsistencies and are the subject of ongoing work.

FIG 1.

Near-complete 16S rRNA gene sequence (>1,450 bp) phylogenetic tree of M. talmoniae clinical isolates and rapidly and slowly growing mycobacteria. The evolutionary distances were computed using the number of differences method (15) and are expressed in the units of the number of base differences per sequence. The strain relationships are based on neighbor-joining and complete deletion analyses.

FIG 2.

Phylogenetic tree of RNA polymerase subunit beta (rpoB) region V gene of M. talmoniae clinical isolates and rapidly and slowly growing mycobacteria. The evolutionary distances were computed using the number of differences method (15) and are expressed in the units of the number of base differences per sequence. The strain relationships are based on neighbor-joining and complete deletion analyses.

FIG 3.

Heat shock protein 65 (hsp65) gene sequence phylogenetic tree of M. talmoniae clinical isolates and rapidly and slowly growing mycobacteria. The evolutionary distances were computed using the number of differences method (15) and are expressed in the units of the number of base differences per sequence. The strain relationships are based on neighbor-joining and complete deletion analyses.

The newly sequenced isolate was compared to the draft genome of the M. talmoniae ATCC BAA-2683^T (GenBank accession number PPEA00000000) using the average nucleotide identity (ANI) method (21); the ANI between the genomes was 99.85%. Using current recommendations of an ANI cutoff of 95% to 96% for a species-level identification criterion (22), the data indicate that these are the same species.

Using the current CLSI guidelines (8) for interpretation of antimicrobial susceptibility testing, the three clinical isolates in the current study and the type strain were susceptible to amikacin, clarithromycin, rifabutin, and trimethoprim-sulfamethoxazole (TMP-SMX). An MIC for TMP-SMX was not available for the type strain. All four isolates were resistant to ciprofloxacin, linezolid, and doxycycline or minocycline (minocycline and/or doxycycline MICs were not performed on the type strain or on the case 1 isolate). All four isolates were intermediate or susceptible to moxifloxacin. The three clinical isolates were either intermediate or resistant to ethambutol. Three of the four isolates were also susceptible to rifampin (MIC, 1 µg/ml), with the type strain resistant at an MIC of 2 µg/ml.

DISCUSSION

The previous and current clinical isolates of M. talmoniae are shown in Table 1, including two previous isolates reported by Davidson et al. (7). Both isolates were derived from respiratory samples, but limited clinical data were available, including the presence or absence of underlying bronchiectasis or drug therapy.

In the first patient, multiple sputum samples were AFB smear and culture positive for M. talmoniae. No other mycobacterial species was recovered at the time. The conversion of her sputum culture to negative occurred within a month after treatment for M. talmoniae was begun. Her symptoms and the improved clinical presentation after beginning therapy for this strain, along with the absence of other mycobacteria or bacterial respiratory pathogens in her cultures at that time, suggest that M. talmoniae was responsible for her clinical disease infection during 2008. This is the first clinical case reported by this new species.

Mycobacterium talmoniae is a potential pulmonary pathogen that should be considered in the differential diagnosis of NTM lung disease. The organism should be differentiated from other slowly growing pulmonary pathogens, such as Mycobacterium avium complex and Mycobacterium kansasii, as noted in the cases presented here, and can currently be identified only by gene sequencing. It is known that due to its unique phylogenetic position (7), M. talmoniae will successfully be identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry, once included in a mycobacterial database.