Inaccuracy of Single-Target Sequencing for Discriminating Species of the <i>Mycobacterium abscessus</i> Group


We determined nucleotide sequences of rpoB, hsp65, and sodA in 59 clinical isolates (from 58 patients) of the Mycobacterium abscessus group. Identification to the species level, based on three target genes, was concordant for 44 isolates (25 M. abscessus, 13 Mycobacterium massiliense, and 6 Mycobacterium bolletii isolates) and discordant for 15 isolates which had "interspecific composite patterns." Sequence analysis of five housekeeping genes also showed composite patterns in 8 of these 15 isolates. 

Mycobacterium abscessus is a rapidly growing mycobacterium (RGM) causing a wide spectrum of disease in humans, including pulmonary disease, skin and soft tissue disease, and disseminated disease (8). It is a major pathogen in patients with cystic fibrosis (CF) (15,23,25,29), in which case it is responsible for severe lung disease and may cause disseminated infection following transplantation (10). M. abscessus has undergone many taxonomic changes since its first description by Moore and Frerichs in 1953, in which this organism was reported to be morphologically and biochemically different from the hitherto known RGM and was named Mycobacterium abscessus (M. abscessus strain ATCC 19977 T ) (22). In 1972, Stanford et al. reported another RGM differing from M. abscessus by only a few characters. The two RGMs were then classified as one species (Mycobacterium chelonei) with two subspecies-M. chelonei subsp. chelonei and M. chelonei subsp. abscessus (formerly M. abscessus) (19,32). In 1984, the species name was changed from M. chelonei to Mycobacterium chelonae (16). This taxonomy remained valid until the beginning of the 1990s, after which DNA/DNA hybridization showed that M. chelonae subsp. abscessus and M. chelonae subsp. chelonae constituted two different species (M. abscessus and M. chelonae) (20). Further development of molecular identification methods in the late 1990s led to a new classification (34). Using different targets, mainly hsp65 and rpoB, it soon became obvious that isolates of M. abscessus were relatively heterogeneous (2,26,31). On the basis of rpoB sequence data, a new species, Mycobacterium massiliense, was identified within the M. abscessus group in 2004, followed by another new species, Mycobacterium bolletii, in 2006 (1,5). Thus, M. abscessus (M. abscessus sensu lato) is now divided into three species, M. abscessus sensu stricto, M. massiliense, and M. bolletii. 
We recently isolated an RGM strain which was identified as M. abscessus sensu stricto (for reasons of simplicity, M. abscessus sensu stricto will hereinafter be referred to as M. abscessus) on the basis of the rpoB sequence but as M. massiliense on the basis of the hsp65 sequence (strain AP3). This suggested the existence of isolates with "interspecific composite patterns" (e.g., isolates with an M. abscessus rpoB sequence and an M. massiliense hsp65 sequence) within the M. abscessus group, potentially leading to inaccuracy for diagnostic approaches based on single-target sequencing. We investigated this issue by determining the sequences of rpoB, hsp65, and another widely used molecular target, sodA (3,18), in a large panel of M. abscessus sensu lato strains. 
The studied panel included 59 clinical isolates of M. abscessus sensu lato obtained from 58 CF patients in France between 1997 and 2007. Strains were grown on sheep blood agar at 37°C for 4 days to obtain visible colonies. Smooth (S) and rough (R) phenotypes were determined as described previously (9). A loopful of colonies was used for DNA extraction by using Tris-EDTA, lysozyme, and proteinase K, in the presence of thiourea to avoid DNA degradation (37). The strains M. abscessus CIP 104536 T (same as ATCC 19977 T ), M. massiliense CIP 108297 T , and M. bolletii CIP 108541 T were included for control purposes. In all strains, hsp65 (441 bp [26]), rpoB (723 bp [3]), and sodA (541 bp [3]) were amplified by PCR using AmpliTaq Gold polymerase (Applied Biosystems, Courtaboeuf, France). Dideoxy sequencing was carried out on both strands with the BigDye Terminator cycle sequencing kit (Applied Biosystems). Sequencing products were purified by gel filtration (Bio-Gel P-100; Bio-Rad, Marnes-la-Coquette, France) and were run on a 3700 DNA analyzer (Applied Biosystems). 
Species identification based on rpoB, hsp65, and sodA sequencing was concordant for 44 isolates (25 M. abscessus [16 S and 9 R], 13 M. massiliense [8 S and 5 R], and 6 M. bolletii [4 S and 2 R] isolates) and discordant for 15 isolates (8 S and 7 R). In 8 of these 15 isolates (see Table 2), both rpoB and hsp65 were 100% identical to the M. abscessus type strain sequence, whereas sodA shared the highest identity with the M. bolletii (isolates 7, 48, 59, 70, AP2, AP6, and AP11) or M. massiliense type strain sequence (isolate 31). In three other isolates, rpoB was most similar to that of M. abscessus (isolates R3, AP3, and 63), whereas both hsp65 and sodA were 100% identical to those of M. massiliense. For isolates 6, 47, 19, and 38, comparisons of rpoB, hsp65, and sodA with the control sequences yielded contradictory data, resulting in the identification of M. massiliense (99.7 to 100% identity) or M. bolletii (99.8 to 100% identity), depending on the target gene. 
The 15 isolates showing "interspecific composite patterns" were further studied by partially sequencing the housekeeping genes argH (argininosuccinate lyase), glpK (glycerol kinase), murC (UDP-N-acetylmuramate-L-Ala ligase), gnd (6-phosphogluconate dehydrogenase), and cya (adenylate cyclase). The nucleotide sequences of these five genes were obtained from GenBank (27) for M. abscessus CIP 104536 T and were determined using the primers presented in Table 1 for M. massiliense CIP 108297 T and M. bolletii CIP 108541 T ; these sequences were submitted to GenBank (accession numbers FJ609780, FJ609781, FJ609782, FJ609783, and FJ609784 for argH, glpK, murC, gnd, and cya of M. bolletii and FJ609785, FJ609786, FJ609787, FJ609788, and FJ609789 for argH, glpK, murC, gnd, and cya of M. massiliense, respectively). The proteins encoded by argH, gnd, and cya have been used for multilocus enzyme electrophoresis analysis of Mycobacterium tuberculosis and the Mycobacterium avium-Mycobacterium intracellulare complex (14, 30), whereas murC and glpK are known housekeeping genes of M. tuberculosis and Mycobacterium leprae (21,28). Dideoxy sequencing was carried out on both strands as described above. 
Sequence data obtained with argH, glpK, murC, gnd, and cya were concordant in seven isolates (three S and four R; all five genes had the highest identity to the same species) and were discordant (four or more genes had the highest identity to the same species) in eight isolates (five S and three R) (Table 2). Among the seven isolates with concordant data, four shared the strongest identity with M. abscessus (isolates 48, AP2, AP6, and AP11) and three with M. massiliense (isolates R3, 6, and 47). These results were consistent with sequence data obtained with hsp65 in all seven isolates, with rpoB in five isolates (48, AP2, AP6, AP11, and 6), and with sodA in only two isolates (R3 and 47). Among the eight isolates showing composite patterns, six could be presumptively identified to the species level (same species assignment provided by two genes among rpoB, hsp65, and sodA and by four genes among argH, glpK, murC, gnd, and cya)-three as M. abscessus (isolates 7, 59, and 70), two as M. massiliense (isolates AP3 and 63), and one as M. bolletii (isolate 38). Finally, two isolates had a highly complex pattern (isolates 19 and 31) that did not allow species identification. 
All isolates were tested for their susceptibility to clarithromycin and minocycline, using the broth microdilution method (Sensititre RGMYCO; Biocentric, Bandol, France) (24). All but 4 of the 44 isolates with concordant rpoB, hsp65, and sodA sequences had MICs to clarithromycin of Յ2 mg/liter. The remaining four isolates (two M. abscessus, one M. massiliense, and one M. bolletii) had MICs of Ͼ32 mg/liter. Thus, in contrast to previous reports (4), most of the M. bolletii isolates studied (five of six) were susceptible to clarithromycin. All but one of the M. abscessus and M. bolletii isolates with concordant rpoB, hsp65, and sodA sequences had MICs to minocycline of Ն16 mg/liter, while 7 of the 13 M. massiliense isolates had MICs of Յ8 mg/liter, including four isolates with MICs of Յ1 mg/liter. The MICs to clarithromycin and minocycline of the 15 strains with "interspecific composite patterns" are shown in Table 2. The MICs to clarithromycin were Յ2 mg/liter for nine isolates and Ն16 mg/liter for five isolates (one was presumptively identified as M. abscessus, two as M. massiliense, one as M. bolletii, and one was unknown). All but one of the isolates (isolate 47, which had a MIC of 8 mg/liter and was presumptively identified as M. massiliense) had MICs to minocycline of Ն32 mg/liter. 
Today, the main method for identifying an RGM to the species level is based on the sequencing of one gene in particular, rpoB, which has been widely studied (1,2). Our study shows that this strategy is not suitable for identifying species within the M. abscessus group, due to the significant number of isolates displaying a composite genetic structure-more than a quarter of the isolates in our series. This relatively high number does not seem to reflect a bias in selection; indeed, we (36). These findings, similar to our own observations, are unlikely to be related to mutations (the presence of specific genetic signatures). Rather, they suggest genetic exchange among members of the M. abscessus group, most probably leading to legitimate recombination events between homologous genes. As shown for the first time here, these events also involve housekeeping genes, such as cya and glpK, and are undoubtedly relatively frequent in this group of bacteria. Certain M. abscessus isolates thus seem to have a composite genetic structure, resulting from genetic exchange between the members of this group. The practical implication of these results is that an M. abscessus sensu lato isolate cannot be reliably identified to the species level by sequencing a single genetic locus, whatever it is. As reported in the study by Devulder et al. (13), sodA was the most variable gene in our study; in 10 composite strains with concordant rpoB and hsp65 sequences, the sodA sequence diverged, and in five of these strains, sodA was the only one that diverged. However, rpoB and hsp65, the genes most frequently used for RGM identification (11-13, 18, 35), also gave diverging data for a number of strains. For two of our isolates (isolates 19 and 31), the sequence data for the eight genes studied, comprising ϳ4,330 bp, did not allow us to distinguish between M. abscessus, M. massiliense, and M. bolletii. 
Furthermore, our findings, together with those of Viana-Niero et al. (35), Kim et al. (18) and Zelazny et al. (36), question the distinction between M. abscessus, M. massiliense, and M. bolletii within the M. abscessus group. M. massiliense and M. bolletii were identified as novel species on the basis of Ͼ3% divergence of their rpoB gene sequence from the sequences of the M. abscessus type strain (1,5,7,36). The difference between M. abscessus and M. massiliense or M. bolletii rpoB nucleotide sequences is indeed about 3%, but there is only a 1.5% difference between those of M. massiliense and M. bolletii. Our results do not call into question the existence of these three entities within the M. abscessus group; indeed, it is clear that composite strains arose from exchanges between these three entities. But the definition of species based on just one or two gene sequences should be questioned (17). Further studies are needed to determine whether these three bacterial entities should be considered truly distinct species within the M. abscessus group. 
Nucleotide sequence accession numbers. The sequences of the following genes were submitted to GenBank: argH, glpK, murC, gnd, and cya of M. bolletii CIP 108541 T under accession numbers FJ609780, FJ609781, FJ609782, FJ609783, and FJ609784, respectively, and argH, glpK, murC, gnd, and cya of M. massiliense CIP 108297 T under accession numbers FJ609785, FJ609786, FJ609787, FJ609788, and FJ609789, respectively. 

TABLE 1 .

 1 Primers used for PCR and sequencing Niero et al. reported two M. bolletii isolates with a sodA sequence identical to that in M. massiliense (35). Kim et al. reported two isolates from South Korea which were identified as M. massiliense with rpoB and sodA and as M. abscessus with hsp65 (18). Very recently, Zelazny et al. reported 7 out of 42 clinical isolates showing ambiguous identification by partial sequencing of rpoB, hsp65, and secA 


	Gene	Primer	Sequence	Amplified fragment size (bp)	Temp (°C)	Reference
	rpoB	MYCOF	5Ј-GGCAAGGTCACCCCGAAGGG-3Ј	723	68	3
		MYCOR	5Ј-AGCGGCTGCTGGGTGATCATC-3Ј		68	
	hsp65	TB11	5Ј-ACCAACGATGGTGTGTCCAT-3Ј	441	60	33
		TB12	5Ј-CTTGTCGAACCGCATACCCT-3Ј		62	
	sodA	SODLGF	5Ј-GAAGGAATCTCGTGGCTGAATAC-3Ј	541	68	3
		SODLGR	5Ј-AGTCGGCCTTGACGTTCTTGTAC-3Ј		70	
	argH	ARGHF	5Ј-GACGAGGGCGACAGCTTC-3Ј	629	60	This study
		ARGHSR1	5Ј-GTGCGCGAGCAGATGATG-3Ј		58	
	glpK	GLPKSF1	5Ј-AATCTCACCGGCGGTGTC-3Ј	609	58	This study
		GLPKSR2	5Ј-GGACAGACCCACGATGGC-3Ј		60	
	murC	MURCSF1	5Ј-CGGACGAAAGCGACGGCT-3Ј	607	60	This study
		MURCSR2	5Ј-CCAAAACCCTGCTGAGCC-3Ј		58	
	gnd	GNDF	5Ј-GTGACGTCGGAGTGGTTGG-3Ј	634	62	This study
		GNDSR1	5Ј-CTTCGCCTCAGGTCAGCTC-3Ј		62	
	cya	ACF	5Ј-GTGAAGCGGGCCAAGAAG-3Ј	647	58	This study
		ACSR1	5Ј-AACTGGGAGGCCAGGAGC-3Ј		60	

TABLE 2 .

 2 Isolates with "composite" gene patterns identified in this study Reference strains used for comparison were M. abscessus CIP 104536 T (same as ATCC 19977 T ), M. massiliense CIP 108297 T , and M. bolletii CIP 108541 T . 


	Isolate a	S/R morphotype	MIC (mg/liter) b CLA MNO	Species c	rpoB hsp65 sodA	% Identity d argH glpK	murC	gnd	cya	Presumptive identification e
	7 (Nantes, 2004)	R	Ͼ32	Ͼ32 M. abscessus	100	100	97.9 98.4 100	99.4 99.4 97.9 M. abscessus
					M. massiliense	96.6 98.3 98.1 95.2 98.1 98.2 97.7 99.8
					M. bolletii	95.9 97.9 100	96.2 98.6 96.7 95.9 97.1
	48 (Pessac, 2004)	S	4	Ͼ32 M. abscessus	100	100	97.6 98.8 99.8 99.3 98.9 99.6 M. abscessus
					M. massiliense	96.2 98.3 97.8 96.2 97.9 97.9 97.4 98.1
					M. bolletii	95.4 97.9 99.2 97.0 98.4 96.3 95.6 97.7
	59 (Paris, 2000)	R	1	Ͼ32 M. abscessus	100	100	97.7 98.2 100	99.6 99.6 97.3 M. abscessus
	70 (Vannes, 2002)	S	2	32 M. massiliense	96.3 98.2 97.9 95.0 98.1 98.3 97.9 97.3
					M. bolletii	95.5 97.9 99.8 96.0 98.6 96.9 96.4 99.2
	AP2 (Paris, 2005)	R	Յ0.12 Ͼ32 M. abscessus	100	100	97.8 98.2 100	99.6 99.6 100	M. abscessus
	AP6 (Paris, 2007)	R	Յ0.12	32 M. massiliense	96.5 98.5 98.0 95.0 98.1 98.3 97.9 98.1
	AP11 (Paris, 2005)	S	0.25	32 M. bolletii	95.9 98.2 99.8 96.0 98.6 96.9 96.2 98.1
	31 (Montpellier, 2004)	S	2	Ͼ32 M. abscessus	100	100	99.4 95.8 98.0 98.0 97.3 99.6 ?
					M. massiliense	96.5 98.5 100	99.8 100	100	99.4 98.1
					M. bolletii	95.7 98.2 98.1 97.4 98.9 96.7 96.7 97.7
	R3 (Roscoff, 2002)	R	Յ0.12 Ͼ32 M. abscessus	100	98.5 99.5 95.6 98.0 99.6 97.6 98.1 M. massiliense
					M. massiliense	96.6 100	100	99.8 100	98.3 100	100
					M. bolletii	95.8 99.1 98.2 97.0 98.9 97.0 96.9 97.3
	AP3 (Boulogne, 2007)	S	Յ0.12 Ͼ32 M. abscessus	99.4 98.2 99.5 95.6 98.0 97.8 97.7 97.9 M. massiliense
					M. massiliense	97.2 100	100	100	100	99.8 99.8 97.9
	63 (Tours, 2004)	S	Յ0.12 Ͼ32 M. bolletii	96.2 98.8 98.2 97.2 98.9 96.5 97.2 99.4
	6 (Nantes, 2003)	R	Ͼ32	32 M. abscessus	96.3 98.5 97.8 95.6 97.9 97.8 97.6 97.7 M. massiliense
					M. massiliense 100	100	98.0 99.6 99.8 99.1 99.6 99.6
					M. bolletii	98.2 99.1 99.8 97.2 98.8 96.9 96.9 96.9
	47 (Grenoble, 2004)	S	Ͼ32	8 M. abscessus	99.7 98.2 99.4 95.6 97.7 98.0 97.3 97.7 M. massiliense
					M. massiliense	98.4 100	100	99.6 99.6 100	99.4 99.6
					M. bolletii	100	99.1 98.2 97.2 98.6 96.7 96.7 96.9
	19 (Paris, 2004)	R	16	Ͼ32 M. abscessus	95.7 98.2 99.6 96.6 100	98.0 95.9 99.4 ?
					M. massiliense	98.3 99.1 100	97.2 98.0 99.6 97.5 98.6
					M. bolletii	99.8 100	98.2 100	98.6 97.0 98.6 97.9
	38 (La Réunion, 2005)	S	Ͼ32	Ͼ32 M. abscessus	95.7 98.2 97.7 96.6 99.1 95.8 95.7 98.1 M. bolletii
					M. massiliense	98.5 99.7 98.2 97.2 98.2 97.0 96.9 97.3
					M. bolletii	100	99.4 100	100	98.8 100	100	100

a Isolate no. (city in France and year of collection). b CLA, clarithromycin; MNO, minocycline. c d Values shown in boldface type indicate the highest percentage of identity. e ?, unknown.
Edouard Macheras, Anne-Laure Roux, Fabienne Ripoll, Valérie Sivadon-Tardy, Cristina Gutierrez, Jean-Louis Gaillard, Beate Heym
Inaccuracy of Single-Target Sequencing for Discriminating Species of the <i>Mycobacterium abscessus</i> Group