Abstract
“Mycobacterium avium subsp. hominissuis” is an important cause of pulmonary disease. It is acquired from environmental sources, but there is no methodology for large population studies. We evaluated the potential of variable-number tandem-repeat (VNTR) analysis. Clinical and household biofilm M. avium isolates underwent molecular identification. Testing for IS901 was done to separate M. avium subsp. avium from M. avium subsp. hominissuis. VNTR types were defined using VNTR loci, and subtyping was performed using 3′ hsp65 and internal transcribed spacer (ITS) sequencing. Forty-nine VNTR types and eight subtypes of M. avium subsp. hominissuis (IS901 negative) were identified among 416 isolates of M. avium from 121 patients and 80 biofilm sites. Of those types, 67% were found only among patient isolates, 11% only among household water isolates, and 23% among both. Of 13 VNTR types that included ≥4 patients, the majority (61.5%) represented geographic clustering (same city). Most VNTR types with multiple patients belonged to the same 3′ hsp65 sequence code (sequevar). A total of 44 isolates belonging to four M. avium subsp. hominissuis VNTR types (8%), including three with the rare Mav-F ITS sequence and 0/8 subspecies, produced amplicons with IS901 PCR primers. By sequencing, all 44 amplicons were not IS901 but ISMav6, which was recently observed in Japan but had not been previously described among U.S. isolates. VNTR analysis of M. avium subsp. hominissuis isolates is easier and faster than pulsed-field gel electrophoresis. Seven VNTR loci separated 417 isolates into 49 types. No isolates of M. avium subsp. avium were identified. The distributions of the VNTR copy numbers, the allelic diversity, and the low prevalence of ISMav6 differed from the findings for respiratory isolates reported from Japan.
INTRODUCTION
The species “Mycobacterium avium subsp. hominissuis” is an important cause of chronic lung disease in the setting of bronchiectasis and disseminated disease in patients with AIDS. The species is acquired from environmental sources, with recent studies focusing on household water. It is present worldwide, with all areas of the United States (especially the southern United States) being considered high-risk areas for the disease.
Efforts to compare isolates of M. avium have been difficult. Three subspecies are currently recognized, namely, M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum. A fourth subspecies, M. avium subsp. hominissuis (including human isolates), has also been proposed (1, 2). IS901 is present in all isolates of M. avium subsp. avium but is not present in isolates of M. avium subsp. hominissuis (1); this difference has been used to separate the two subspecies (1, 2). Strain comparisons within M. avium (as well as with other members of the Mycobacterium avium complex [MAC], especially Mycobacterium intracellulare) have most frequently utilized pulsed-field gel electrophoresis (PFGE) (3, 4), which is expensive, technically difficult, time-consuming, and useful only for comparing small numbers of isolates. Other methods, such as repetitive sequence-based PCR (rep-PCR) (5), random amplified polymorphic DNA PCR (RAPD-PCR) (6), gene sequencing (2), and sequencing of the 16S-23S internal transcribed spacer (ITS) region (1, 2), have also been employed for M. avium complex strain comparisons.
Genome sequences of several strains and subspecies of M. avium (including the type strain of M. avium subsp. avium, ATCC 25291) have been made available (7). A number of variable-number tandem repeats (VNTRs) of the minisatellite class (50 to 60 bp) have been identified and used in preliminary studies. Such studies include the use of mycobacterial interspersed repetitive unit (MIRU) loci (8) and M. avium tandem-repeat (MATR) loci (some of which recognize the same tandem repeat) (9). VNTR analysis has been used for strain comparisons and population studies of other species of mycobacteria, including M. intracellulare, Mycobacterium ulcerans, and Mycobacterium tuberculosis (8–18). Studies on the use of VNTR analysis for M. intracellulare isolates were reported from Japan and France in 2009 and 2010 (11, 13), respectively, and from the United States in 2013 (12). Similar studies on the use of VNTR analysis for M. avium isolates have also been reported, but none of those studies evaluated isolates from the United States (9, 15, 18). Here we describe the use of M. avium VNTR analysis for comparison of environmental water and respiratory disease isolates from the United States and utilization of the composite data to create a database of isolates.
MATERIALS AND METHODS
Study design.
There currently is no established, rapid, simple, discriminatory method for M. avium strain comparisons or population studies. Studies have shown the ability of VNTR analysis to meet the criteria for discrimination from the closely related species M. intracellulare (11–13), and preliminary studies have shown VNTR analysis to be feasible with M. avium (8, 9). Thus, we undertook a study on the use of VNTR analysis in the clinical setting of U.S. patients with nodular bronchiectasis and M. avium. Isolates studied for microbiological relapse of MAC-related lung disease were compared with PFGE and VNTR analysis, and VNTR types with two or more patients were compared by 5′ hsp65 sequencing. VNTR typing results for select groups were compared with results from pulsed-field gel electrophoresis (PFGE) and 3′ hsp65 sequencing.
Patients.
Patients were selected from bronchiectasis and nontuberculous mycobacteria (NTM) clinics at the University of Texas Health Science Center at Tyler (UTHSCT) and the Pulmonary and Critical Care Division, Lankenau Medical Center (Wynnewood, PA). Patient isolates were candidates for study if the patients had nodular bronchiectasis. Most patients met current American Thoracic Society (ATS)/Infectious Diseases Society of America (IDSA) 2007 guidelines for clinical disease (19). Some patients and their isolates were included in previous or current clinical studies from the UTHSCT and the Lankenau Medical Center (20). Clinical information on the presence of bronchiectasis and the home address (city) were extracted from medical records. This study was approved by the UTHSCT and Lankenau Medical Center institutional review boards (IRBs).
Isolates.
Patients who had nodular bronchiectasis and whose MAC isolates were submitted for strain comparisons because of microbiological relapse during or after drug treatment of M. avium subsp. hominissuis or for comparisons with household water/biofilm isolates or who had a request for sputum MAC species identification and were found to have M. avium were chosen for study; the latter two groups consisted of only a single M. avium isolate. For patients with suspected microbiological relapse, current isolates were compared to previous isolates stored at −70°C or stored in their original broth culture vials at room temperature following primary isolation. Sputum cultures from patients had been submitted to and processed by the clinical mycobacteriology laboratory as described previously (3, 4, 12). Sputum isolates from three recent studies (20; R. J. Wallace, Jr., unpublished data) comparing patient and household water MAC isolates (5) were also included. Isolates were from Texas, Oklahoma, Louisiana, Arkansas, and Pennsylvania. These studies were also approved by the UTHSCT and Lankenau Medical Center IRBs.
Environmental isolates from household water and biofilms from multiple sites and states were also collected and underwent processing and culture as described previously (5). These isolates were from showerhead filters, shower pipes, bathroom and kitchen faucet filters, bathroom and kitchen pipes, bathtub inlets, and (when available) hot tub filters. Isolates were obtained from Texas, Louisiana, Oklahoma, Maryland, and Pennsylvania.
Species and subspecies identification.
Isolates for study initially were identified as MAC using a commercial hybridization assay (AccuProbe MAC probe; Hologic Inc.) and then were identified as M. avium, M. intracellulare/Mycobacterium chimaera, or MAC X using the multiplex 16S rRNA gene PCR technique described by Wilton and Cousins (21) or hsp65 PCR-restriction analysis (PRA) (22). To confirm the species identification, sequencing of the internal transcribed spacer (ITS) region was performed (23, 24).
IS901 has been shown to be present in all isolates of M. avium subsp. avium and M. avium subsp. silvaticum but absent in isolates of M. avium subsp. hominissuis (1, 2). Therefore, one or more isolates from each patient underwent PCR for IS901, as described previously (2). Clinical isolates with negative results for IS901 by PCR were considered to belong to M. avium subsp. hominissuis. Isolates that produced an amplicon the size of IS901 underwent sequencing of the PCR product, to determine whether the product was indeed IS901. Three reference strains of M. avium subsp. avium, i.e., the type strain ATCC 25291T (TMC 724; chicken, serotype 2), ATCC 35718 (TMC 721; child's lymph node, serotype 3), and ATCC 35712T (TMC 701; chicken, serotype 2), were included as positive controls. Two human reference strains of M. avium subsp. hominissuis (which has no designated type strain), i.e., ATCC 700898 (strain MAC 101) and MAC 104 (both from the blood of patients with AIDS), were included as negative controls.
Pulsed-field gel electrophoresis.
PFGE was performed as described previously, using the restriction enzymes XbaI and AseI (3, 4, 12). Isolates from the same patient were compared and classified as indistinguishable, closely related, possibly related, or unrelated on the basis of differences in band number and position, using methods originally described by Tenover et al. for bacterial outbreaks and then modified for MAC (4, 12, 25). Strains in the first three categories were considered clonal. Different (unrelated) genotypes from the same patient were given letter designations; numbers were added to the letter designations (e.g., A2) if minor band differences were observed.
VNTR analysis.
All isolates with different genotypes (unrelated PFGE patterns) from individual patients with multiple isolates, as well as single isolates that did not undergo PFGE, underwent VNTR analysis. For some patients, multiple isolates of the same PFGE genotype also underwent typing. One MIRU locus (MIRU locus 3) described by Bull et al. (8) and six MATR loci described by Inagaki et al. (9) (MATR-1, MATR-2, MATR-3, MATR-7, MATR-13, and MATR-14) were used; those six loci had the greatest allelic diversity of 15 loci studied by Inagaki et al. (9). One of the MATR loci amplified was the same locus as a MIRU tandem repeat (MIRU tandem repeat 292 = MATR-2) (9). PCR products were sized with 2% agarose gel electrophoresis. Allele designations for each amplicon reflected the number of complete tandem repeats.
DNA fingerprinting of isolates with the same VNTR types, using PFGE and 3′ hsp65 gene sequencing.
M. avium isolates from five or more different patients that were subsequently shown to belong to the same VNTR type were compared to each other by using PFGE for selected VNTR types. Isolates from two or more different patients also underwent comparisons by using 3′ hsp65 gene sequencing, as described previously (2, 26).
PCR and DNA sequencing of ITS, VNTR alleles of different sizes, and IS901.
Sequencing of the ITS region (263 bp) was performed as described previously (23, 24). Results were compared to ITS sequences in GenBank, including the Mav-A (GenBank accession no. EF521901.1), Mav-B (GenBank accession no. CP000479.1), Mav-G (GenBank accession no. AF315839.1), and Mav-F (GenBank accession no. AF315838) sequences. Examples of each of the alleles of different sizes for each of the seven primers were also sequenced, to determine the length (size) of each tandem repeat.
As noted previously, isolates that were PCR positive for IS901 underwent amplicon sequencing. The sequences then underwent BLAST analysis (www.ncbi.nlm.nih.gov/BLAST) using the GenBank database.
Comparison of VNTR copy numbers and allelic diversity with isolates from Japan.
A comparison of the VNTR copy numbers and allelic diversity for each of the MATR-VNTR loci used to separate the VNTR types from the United States in the current study was made with a prior study by Inagaki et al. of respiratory isolates from Nagoyi, Aichi, Japan (9). The allelic diversity among the U.S. isolates was calculated using the formula of Selander et al. (27).
RESULTS
Patients and isolates.
A total of 416 isolates of M. avium subsp. hominissuis from 121 patients and 80 household environmental biofilm sites, three reference strains of M. avium subsp. avium (one patient strain and two chicken strains), and two reference strains of M. avium subsp. hominissuis were characterized. Each different VNTR genotype was counted only once per patient, although multiple patients had more than one VNTR type during the follow-up period (and were counted more than once), and multiple isolates of the same VNTR types (range, 2 to 21 isolates) contributed to the total of 416 isolates. Patients had typical radiographic findings of nodular bronchiectasis and MAC disease. Additionally, most patients met the 2007 American Thoracic Society/Infectious Disease Society of America guidelines for NTM lung disease (19). Two exceptions were reference strains MAC 104 and ATCC 700898, which were blood isolates recovered from patients with AIDS.
Species and subspecies identification.
PCR for IS901 yielded positive results for the three reference strains of M. avium subsp. avium (ATCC 25291T and ATCC 35712, both from chickens, and ATCC 35718T, from a human cervical lymph node) (1, 2). The IS901 PCR also produced an amplicon of the same size as IS901 for four clinical VNTR types (type 8, which contained one isolate from one patient; type 40, which contained five isolates from two patients; type 37a, which contained two isolates from one patient; and type 51, which contained 36 isolates from three environmental sources and 11 patients) containing a total of 44 isolates from 15 patients (Fig. 1). PCR for IS901 yielded negative results (did not produce an amplicon) for all of the remaining clinical and environmental isolates and the two reference strains of M. avium subsp. hominissuis (strains MAC 104 and ATCC 700898) (data not shown.).
FIG 1.
PCR analysis of clinical isolates of M. avium subsp. hominissuis using IS901 primers. (A) Analysis of clinical isolates. Lane 1, 100-bp ladder; lane 2, M. avium subsp. avium type strain ATCC 25291; lanes 3 to 12, clinical isolates of M. avium subsp. hominissuis from different VNTR groups; lane 13, PCR water control. (B) Analysis of Mav-F isolates. Lane 1, 100-bp ladder; lane 2, ATCC 25291T; lanes 3 and 4, VNTR type 40 isolates; lanes 5 to 8, VNTR type 51 isolates from four patients; lane 9, PCR water control. Numbers on the left, marker sizes (in base pairs). As expected, ATCC 25291T produced an amplicon with these primers (panels A and B, lanes 2), consistent with the presence of IS901. One isolate from VNTR type 40 and one isolate from VNTR type 51 also generated bands with sizes similar to that of the IS901 amplicon (panel A, lanes 8 and 12, respectively). These results were confirmed using additional isolates from the two VNTR types (panel B, lanes 3 to 8). With sequencing, the PCR products from all isolates except for the M. avium subsp. avium type strain ATCC 25291 showed 100% sequence identity to ISMAV6 but not IS901.
PFGE.
Multiple MAC isolates from patients suspected of microbiological relapse underwent species or subspecies identification (including isolates obtained before and after the suspected relapse); the test isolates underwent PFGE, and M. avium isolates underwent VNTR typing. Single isolates of M. avium subsp. hominissuis from patients not suspected of relapse did not undergo PFGE analysis. A total of 81 isolates of M. avium subsp. hominissuis from 28 patients with suspected microbiological relapse underwent simultaneous VNTR typing and PFGE analysis. Ten patients had multiple different M. avium VNTR types at different times (mean, 2.4 types per patient [range, 2 to 6 types per patient]). All different VNTR types from the same patients had unrelated (different) PFGE patterns.
Seventeen patients had multiple sputum isolates of the same VNTR type at different times (mean, 3.4 isolates per patient [range, 2 to 6 isolates per patient]). A total of 14 of those patients had 42 compared isolates, with all of the isolates from each patient having indistinguishable PFGE patterns. A total of 9 patients had 10 isolates with related (clonal) PFGE patterns, compared to the predominant (most frequently occurring) PFGE clone, and 4 patients had 5 isolates with the same M. avium VNTR type as ≥1 isolate but unique (unrelated) PFGE patterns. Some patients had both indistinguishable and clonal patterns; therefore, the patient number was >17.
PFGE comparisons of M. avium with multiple other isolates of different MAC species (M. intracellulare, M. chimaera, or other MAC X species) from the same patient were also made for those 28 patients. All M. avium PFGE profiles were unique with respect to those isolates as well.
VNTR analysis.
Multiple M. avium strains with different genotypes from the same patients that had undergone PFGE (range, 2 to 4 genotypes) and single isolates that had not undergone PFGE underwent VNTR typing, as did the five reference strains. Isolates from the same patients that were considered clonal on the basis of PFGE results had the same VNTR type. All genotypes of M. avium produced satisfactory VNTR amplicons for gel sizing and DNA sequencing except for three loci in single isolates, which resulted in no PCR product. Amplicons of indeterminate copy number were confirmed by sequencing. Examples of five amplicons of different sizes, with different numbers of complete tandem repeats for MATR-7, are shown in Fig. 2.
FIG 2.
Examples of different amplicon sizes associated with different tandem-repeat copy numbers for isolates of M. avium subsp. hominissuis and MATR-7 primers. Gel electrophoresis was performed with a 2% agarose gel in 1× Tris-acetate-EDTA (TAE) buffer. Lane 1, 500-bp amplicon with 5 complete copies (MA-4214); lane 2, 450-bp amplicon with 4 complete copies (MA-4826); lane 3, 395-bp amplicon with 3 complete copies (ATCC 700898); lane 4, 320-bp amplicon with 2 complete copies (MA-4616); lane 5, 290-bp amplicon with 1 complete copy (MA-5029); lane 6, 100-bp ladder.
The different tandem-repeat copy numbers and amplicon sizes observed for each of the seven primers were given allele numbers that matched the numbers of complete tandem repeats (partial tandem repeats were excluded). Amplicons from each primer set were grouped together as a single allele if their size differences (generally <20 bp) did not allow separation on the sizing gel (Table 1). This resulted in 49 VNTR types of M. avium subsp. hominissuis (including the two reference strains) and three VNTR types of the three reference strains of M. avium subsp. avium (VNTR types 24, 29, and 43) (Table 2). Eight VNTR subtypes (with a number followed by a letter) were isolates that had the same tandem-repeat copy numbers for all seven primer sets but different ITS sequences (i.e., 14 and 14a; 15 and 15a; 27 and 27a; 31, 31a, and 31b; 36 and 36a; 37 and 37a; and 38 and 38a). The 49 VNTR types differed by one or more tandem-repeat copy numbers. Most VNTR types of M. avium subsp. hominissuis included a single genotype from one patient (33/49 types [67.3%]). Only 13 VNTR types and subtypes included isolates from four or more different patients (range, 4 to 14 patients). Eight of these 13 groups (61.5%) had geographic clustering (same town), while five did not, based on home addresses (Table 3).
TABLE 1.
VNTR tandem-repeat size, copy number, amplicon size, and assigned allele number for isolates of M. avium subsp. hominissuis, with seven tandem-repeat loci (8, 12)
| Locus, amplicon size, and copy no.a | 5 alleles | 4 alleles | 3 alleles | 2 alleles | 1 allele | 0+ alleles | 0 alleles |
|---|---|---|---|---|---|---|---|
| MIRU locus 3 (53 bp) | |||||||
| Amplicon size (bp) | 460b | 400 | 350 | 300 | 260 | ND | ND |
| Copy no. | 5.2b | 4.2 | 3.2 | 2.2 | 1.2 | ND | ND |
| MATR-7 (57 bp) | |||||||
| Amplicon size (bp) | 500 | 450 | 395b | 330 | 280 | ND | ND |
| Copy no. | 5.1 | 4.1 | 3.1b | 2.1 | 1.1 | ND | ND |
| MATR-1 (53 bp) | |||||||
| Amplicon size (bp) | 490 | 430 | ND | 330b | 285 | ND | ND |
| Copy no. | 5.4 | 4.4 | ND | 2.4b | 1.4 | ND | ND |
| MATR-2 (53 bp) | |||||||
| Amplicon size (bp) | ND | ND | 350 | 300b | 250 | 195 | ND |
| Copy no. | ND | ND | 3.1 | 2.1b | 1.1 | 0.36 | ND |
| MATR-3 (53 bp) | |||||||
| Amplicon size (bp) | 460b | 400 | 350 | 300 | 230 | ND | ND |
| Copy no. | 5.2b | 4.2 | 3.2 | 2.2 | 1.2 | ND | ND |
| MATR-13 (56 bp) | |||||||
| Amplicon size (bp) | ND | ND | ND | ND | 340b | 295 | 240 |
| Copy no. | ND | ND | ND | ND | 1.9b | 0.9 | 0 |
| MATR-14 (58 bp) | |||||||
| Amplicon size (bp) | ND | 450b | 450 | 395 | 335 | 280c | ND |
| Copy no. | ND | 3.7b | 3.7 | 2.7 | 1.7 | 0.7c | ND |
Copy numbers were rounded to the nearest tenth. The tandem-repeat allele numbers were determined by the numbers of complete repeats. The amplicon sizes include the flanking regions and the tandem repeats. The sizes of the complete tandem repeat are indicated in parentheses. ND, not detected.
Size of the tandem repeat of M. avium subsp. hominissuis reference strain MAC 104.
Seen only with the M. avium subsp. avium reference strain.
TABLE 2.
VNTR types among M. avium subsp. hominissuis isolates from environmental sources and patients with nodular bronchiectasis, determined using six MATR loci described by Inagaki et al. (9) and one MIRU VNTR locus described by Bull et al. (8)
| VNTR typea | Copy no. |
No. of isolates |
ITS sequence | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| MIRU locus 3 | MATR-7 | MATR-1 | MATR-2 | MATR-3 | MATR-13 | MATR-14 | From patients | From environmental sources | Total tested | ||
| 1 | 5 | 5 | 2 | 0 | 5 | 1 | 1 | 1 | 2 | 3 | Mav-A |
| 2 | 5 | 5 | 1 | 0 | 5 | 1 | 1 | 0 | 2 | 2 | Mav-A |
| 3 | 5 | 4 | 2 | 2 | 5 | 1 | 1 | 6 | 13 | Mav-B | |
| 3a | 5 | 4 | 2 | 2 | 5 | 1 | 1 | 1 | 3 | Mav-B | |
| 4 | 5 | 4 | 2 | 2 | 5 | 0+ | 1 | 3 | 4 | Mav-A | |
| 5 | 5 | 4 | 2 | 2 | 2 | 1 | 1 | 1 | 1 | Mav-B | |
| 6 | 5 | 4 | 2 | 2 | 3 | 0+ | 2 | 1 | 1 | Mav-B | |
| 7 | 5 | 4 | 2 | 0 | 2 | 1 | 1 | 1 | 1 | Mav-B | |
| 8 | 5 | 4 | 2 | 1 | 5 | 1 | 1 | 1 | 1 | Mav-B | |
| 9 | 5 | 3 | 2 | 3 | 5 | 1 | 1 | 1 | 1 | NA | |
| 10 | 5 | 3 | 2 | 2 | 5 | 1 | 3 | 2b (M. avium subsp. hominissuis blood isolates MAC 104 and ATCC 700898) | 3 | Mav-B | |
| 11 | 5 | 3 | 2 | 2 | 5 | 1 | 1 | 1 | 1 | Mav-A | |
| 12 | 5 | 3 | 2 | 1 | 5 | 1 | 1 | 1 | Mav-B | ||
| 13 | 5 | 3 | 2 | 0 | 5 | 1 | 2 | 1 | 5 | 9 | Mav-A |
| 14 | 5 | 3 | 2 | 0 | 5 | 1 | 1 | 1 | 1 | Mav-B | |
| 14a | 5 | 3 | 2 | 0 | 5 | 1 | 1 | 9b | 22 | 30b | Mav-A |
| 15 | 5 | 2 | 2 | 0 | 5 | 1 | 1 | 1 | 3 | Mav-B | |
| 15a | 5 | 2 | 2 | 0 | 5 | 1 | 1 | 1 | 1 | Mav-A | |
| 16 | 5 | 1 | NP | 0 | 2 | 1 | 1 | 1 | 1 | Mav-A | |
| 17 | 5 | 1 | 1 | 0 | 5 | 1 | 1 | 0 | 1 | 1 | Mav-B |
| 18 | 4 | 3 | 2 | 2 | 5 | 1 | 3 | 2 | 2 | Mav-B | |
| 19 | 4 | 3 | 2 | 2 | 4 | 1 | 3 | 5 | 1 | 13 | Mav-B |
| 20 | 4 | 3 | 2 | 2 | 4 | 0+ | 3 | 1 | 1 | Mav-B | |
| 21 | 4 | 3 | 2 | 3 | 4 | 1 | 1 | 2 | 1 | Mav-B | |
| 22 | 4 | 3 | 2 | 0 | 4 | 1 | 1 | 1 | 1 | 2 | Mav-B |
| 23 | 4 | 3 | 2 | 0 | 3 | 1 | 1 | 0 | 1 | 1 | Mav-A |
| 24 | 4 | 1 | 1 | 2 | 4 | 0+ | 2 | ATCC 35718 (human) | Mav-A | ||
| 25 | 3 | 5 | 2 | 3 | 3 | 1 | 1 | 1 | 1 | Mav-B | |
| 26 | 3 | 5 | 1 | 0 | 3 | 1 | 1 | 1 | 2 | 8 | Mav-A |
| 27 | 3 | 4 | 2 | 2 | 3 | 1 | 1 | 1 | 3 | Mav-B | |
| 28 | 3 | 3 | 2 | 3 | 3 | 2 | 2 | 1 | 2 | Mav-A | |
| 29 | 3 | 3 | 2 | 3 | 3 | 1 | 2 | ATCC 35712 (chicken) | Mav-B | ||
| 30 | 3 | 3 | 2 | 3 | 3 | 1 | 2 | 4 | 6 | 29 | Mav-B |
| 31 | 3 | 3 | 2 | 3 | 3 | 1 | 1 | 2 | 2 | Mav-A | |
| 31a | 3 | 3 | 2 | 3 | 3 | 1 | 1 | 14b | 6 | 82 | Mav-B |
| 31b | 3 | 3 | 2 | 3 | 3 | 1 | 1 | 1 | 2 | 1 | Mav-G |
| 32 | 3 | 3 | 2 | 2 | 3 | 1 | 1 | 1 | 1 | Mav-B | |
| 33 | 3 | 3 | 2 | 2 | 3 | 0+ | 3 | 2 | 6 | Mav-B | |
| 34 | 3 | 3 | 2 | 2 | 3 | 0+ | 1 | 1 | 1 | Mav-B | |
| 35 | 3 | 3 | 2 | 0 | 4 | 1 | 1 | 0 | 2 | 2 | Mav-B |
| 36 | 3 | 3 | 2 | 0 | 3 | 1 | 2 | 6 | 6 | Mav-A | |
| 36a | 3 | 3 | 2 | 0 | 3 | 1 | 2 | 2b | 7 | 16 | Mav-A |
| 37 | 3 | 3 | 2 | 0 | 3 | 1 | 1 | 9b | 12 | 52 | Mav-A |
| 37a | 3 | 3 | 2 | 0 | 3 | 1 | 1 | 6 | 9 | Mav-B | |
| 38 | 3 | 3 | 1 | 3 | 3 | 1 | 1 | 2 | 1 | 4 | Mav-A |
| 38a | 3 | 3 | 1 | 3 | 3 | 1 | 1 | 0 | 3 | 3 | Mav-B |
| 39 | 3 | 2 | 2 | 3 | 3 | 1 | 1 | 1 | 2 | Mav-B | |
| 40 | 3 | 2 | 2 | 0 | 3 | 1 | 1 | 2 | 5 | Mav-F | |
| 41 | 3 | 1 | 4 | 0 | 3 | 1 | 1 | 1 | 5 | Mav-B | |
| 42 | 3 | 1 | 2 | 2 | 3 | 1 | 1 | 1 | 21 | Mav-A | |
| 43 | 3 | 1 | 1 | 2 | 3 | 0+ | 0 | ATCC 25291T (chicken) | Mav-A | ||
| 44 | 2 | 4 | 2 | 2 | 2 | 1 | 1 | 4 | 6 | Mav-B | |
| 45 | 2 | 3 | 2 | 3 | 3 | 1 | 3 | 1 | 1 | Mav-B | |
| 46 | 2 | 3 | 2 | 2 | 2 | 1 | 3 | 2 | 4 | Mav-B | |
| 47 | 2 | 2 | 1 | 0 | 3 | 0 | 1 | 0 | 1 | 1 | Mav-B |
| 48 | 1 | 4 | 2 | 2 | 2 | 1 | 1 | 1 | 1 | Mav-B | |
| 49 | 1 | 3 | 2 | 2 | 1 | 0 | 1 | 1 | 1 | Mav-B | |
| 50 | 1 | 2 | 2 | 0 | 1 | 1 | 1 | 1 | 2 | Mav-F | |
| 51 | 1 | 1 | 1 | 1 | 1 | 0 | 1 | 1b | 3 | 36b | Mav-F |
| 52 | NP | 4 | 2 | 2 | 2 | 1 | 1 | 6b | 6 | Mav-B | |
Some patients had more than one isolate tested. Isolates with the same VNTR copy numbers but different ITS sequences or different 3′ hsp 65 codes (sequevars) were given the same VNTR number plus a letter (e.g., 3a or 14a). Three reference strains of M. avium subsp. avium and two reference strains of M. avium subsp. hominissuis were controls. VNTR types were validated as M. avium by sequencing of the ITS1 region. The reference strains were as follows: ATCC 25291, type strain for M. avium subsp. avium; ATCC 35718, M. avium subsp. avium; ATCC 35712, M. avium subsp. avium; ATCC 700898, M. avium subsp. hominissuis; strain MAC 104, M. avium subsp. hominissuis. NA, not available; NP, no PCR product (amplicon).
Patients/isolates with geographic clustering.
TABLE 3.
3′ hsp65 sequevars in 22 M. avium VNTR types or ITS subtypes, of which 20 were VNTR types with two or more patients (including outbreak isolates)
| No.a | VNTR type or subtypeb | No. of patients | Code no. | Associated with outbreak | Sequence type |
|---|---|---|---|---|---|
| 1 | 3 | 6 | 1 | ? | |
| 3 | 1 | 2 | − | ||
| 2 | 4 | 3 | 1 | − | |
| 3 | 10 | 4 | 1 | − | |
| 4 | 14a | 10 | 1 | + | |
| 5 | 18 | 2 | NA | − | |
| 6 | 19 | 5 | 3 | − | |
| 7 | 21 | 2 | NA | − | |
| 8 | 30 | 4 | 9 | − | |
| 9 | 31 | 2 | NA | − | |
| 10 | 31a | 14 | 2 | + | |
| 11 | 33 | 6 | 1 | − | |
| 12 | 36 | 5 | 1 | + | |
| 13 | 37 | 7 | 2 | + | |
| 14 | 37a | 4 | 2 | + | |
| 37a | 1 | 9 | − | ||
| 37a | 1 | 1 | − | ||
| 15 | 38 | 2 | NA | − | |
| 16 | 40 | 2 | 2 | − | Mav-F |
| 17 | 42 | 1 | 2 | − | |
| 18 | 44 | 4 | 1 | − | |
| 19 | 46 | 2 | 1 | − | |
| 20 | 50 | 1 | 15 | + | Mav-F |
| 21 | 51 | 11 | 15 | + | Mav-F |
| 22 | 52 | 6 | NA | + |
Arbitrary numerical identifier for type.
Only two VNTR types or subtypes consisted of more than one 3′ hsp65 sequevar (i.e., code numbers), and 6/8 types with five or more patients were part of a known cluster or outbreak. The total includes 20 VNTR types with two or more patient isolates and 2 with single isolates. NA, not available.
DNA fingerprinting of isolates with the same VNTR types, using PFGE and 3′ hsp65 gene sequencing.
PFGE was performed for three VNTR types that included five or more patients or household water sites. PFGE was performed for VNTR type 51, which included three isolates from different patients from the same town and two isolates from other geographic sites (cities) (Fig. 3). Four of the isolates were closely related and one (MA-5511) (Fig. 3, lane 4) was possibly related. By 3′ hsp65 gene sequencing, all five isolates belonged to code 15 (2, 26).
FIG 3.
PFGE of XbaI digests of VNTR type 51 (Mav-F ITS sequence) isolates from five different patients. Lane 1, MA-5327; lane 2, MA-5308; lane 3, MA-4925; lane 4, MA-5295; lane 5, MA-5511; lane 6, ladder. The isolates in lanes 1 and 2 were from patients from different cities, and those in lanes 3 to 5 were from patients from the same city (different from the first two cities). The isolates in lanes 1 and 4 were indistinguishable, those in lanes 2 and 3 were closely related, and that in lane 5 was possibly related (4). The first four isolates and potentially all five isolates were considered clonal. All five isolates belonged to the relatively unusual 3′ hsp65 sequevar code 15 (2, 26). XbaI and a 1% agarose gel were used.
PFGE was also performed on five isolates from four patients with VNTR type 10, including two reference strains (strains MAC 104 and ATCC 700898 from AIDS patients, collected in the same hospital) and two clinical isolates from other cities. The two reference strains were indistinguishable, as were two isolates obtained from the same patient, 6 months apart. Two of the patient isolates were only possibly related, which suggests that this cluster may consist of more than one VNTR type (Fig. 4). By 3′ hsp65 gene sequencing, all five isolates belonged to code 1 (2). PFGE was performed on isolates of a third VNTR type, with all isolates appearing clonal, comparable to the results for VNTR type 51.
FIG 4.
PFGE of XbaI digests of five VNTR type 10 isolates from four different patients, showing that some VNTR types did not consist of patient isolates with indistinguishable or closely related PFGE profiles. Lanes 1 and 9, linear standards; lane 2, strain MAC 104; lane 3, ATCC 700898 (MAC 102); lane 6, strain 07-6464; lanes 4 and 5, two isolates from the same patient, obtained 6 months apart; lane 7, ATCC 25291T (control); lane 8, ATCC 33712 (control). The isolates in lanes 2 and 3 were indistinguishable, while the isolates in lanes 4 and 5 were considered indistinguishable. The isolates in lanes 2, 5, and 6 were considered possibly related. The control strains in lanes 7 and 8 were unrelated. The type 10 isolates all belonged to 3′ hsp65 sequevar code 1 (2).
PCR and DNA sequencing of ITS, IS901 PCR amplicons, and 3′ hsp65. (i) ITS.
Among the 49 M. avium subsp. hominissuis VNTR types, isolates from each patient underwent ITS sequencing. Isolates that had the same VNTR type but different ITS sequences were given the same VNTR number but one was given a letter designation (e.g., 15 and 15a). This analysis revealed that 33 types or subtypes were Mav-B (58.9%), 18 were Mav-A (32.1%), 3 were Mav-F (5.4%), 1 was Mav-G (1.8%), and 1 did not amplify. Other previously described ITS sequences in isolates of M. avium (Mav C, Mav-D, and Mav-E sequences) were not encountered (1, 24, 28). Of the three reference strains of M. avium subsp. avium, ATCC 35712 was surprisingly found to have the Mav-B type, while the remaining two strains, ATCC 35718 and ATCC 25291, had the expected Mav-A type (1).
The three VNTR types that contained Mav-F ITS sequences included 2 patients (VNTR type 40), 1 patient (type 50), and 10 patients (VNTR type 51). All three types had VNTR copy numbers for one or more loci seen infrequently among the other VNTR types (Table 2).
(ii) Insertion sequences.
The three M. avium subsp. hominissuis VNTR types that contained the Mav-F ITS sequence (types 40, 50, and 51) and one that contained the Mav-B sequence (type 8) also produced an amplicon with IS901 PCR primers, along with VNTR type 8 (Mav-B) (a total of 44 isolates in the four VNTR types). Sequencing of the PCR product revealed that it had 100% identity with the recently described ISMav6 from Japan (GenBank accession no. AB447556) (29). Sequencing of the PCR product from the M. avium subsp. avium type strain ATCC 25291 and two other M. avium subsp. avium strains, ATCC 35718 and ATCC 35712, revealed 100% identity with IS901 (GenBank accession no. X59272).
(iii) 3′ hsp65 sequences.
Twenty-two VNTR types, including all those that included two or more different patients, were sequenced (Table 3). The 3′ hsp65 codes (sequevars) identified included codes 1, 2, 3, 4, 9, and 15. All except three patient isolates with the same VNTR type belonged to the same 3′ hsp65 codes (sequevars) (Table 3); this included VNTR types 40 and 51, which were two of the three VNTR types with the Mav-F ITS sequence. The isolates containing the Mav-F ITS sequence were the only isolates studied that belonged to 3′ hsp65 sequence code 15.
Comparison of VNTR copy numbers with isolates from Japan.
A comparison of the number of VNTR types with the same tandem-repeat copy numbers for each of the six MATR-VNTR loci used to separate the 52 VNTR types in the current U.S. study and a prior study of 70 VNTR types among respiratory isolates from Japan (9) is shown in Table 4. M. avium subsp. hominissuis reference strain MAC 104 was included in both studies and showed the same tandem-repeat copy numbers for the same loci in both. These six loci showed the greatest diversity of all among the isolates from Japan, but only three of the loci (MATR-2, MATR-3, and MATR-7) had comparable diversity among the U.S. isolates (Table 4). Overall, the ranges of copy numbers for each locus were similar but the distributions were very different. These findings suggest many of the VNTR types differ between the two geographic areas.
TABLE 4.
Allelic diversity among 51 VNTR types for respiratory isolates from the United States and 70 previously published VNTR types for respiratory isolates from Japan (9)
| VNTR locusa | No. of isolates with complete tandem-repeat no. of: |
Allelic diversity | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | NP | ||
| MATR-1 | ||||||||||
| United States | 0 | 3 | 45b | 0 | 1 | 1 | 0 | 0 | 1 | 0.217 |
| Japan | 1 | 30 | 38b | 0 | 0 | 1 | 0 | 0 | 0.514 | |
| MATR-2 | ||||||||||
| United States | 17 | 3 | 20b | 11 | 0 | 0 | 0 | 0 | 0 | 0.685 |
| Japan | 29 | 34 | 5b | 2 | 0 | 0 | 0 | 0 | 0 | 0.581 |
| MATR-3 | ||||||||||
| United States | 0 | 2 | 7 | 24 | 5 | 13b | 0 | 0 | 0 | 0.684 |
| Japan | 0 | 42 | 10 | 1 | 3 | 14b | 0 | 0 | 0 | 0.581 |
| MATR-7 | ||||||||||
| United States | 0 | 4 | 4 | 30b | 10 | 2 | 0 | 0 | 1 | 0.601 |
| Japan | 0 | 20 | 27 | 3b | 0 | 5 | 14 | 1 | 0 | 0.718 |
| MATR-13 | ||||||||||
| United States | 0 | 7 | 44b | 0 | 0 | 0 | 0 | 0 | 0 | 0.237 |
| Japan | 38 | 3 | 29b | 0 | 0 | 0 | 0 | 0 | 0 | 0.525 |
| MATR-14 | ||||||||||
| United States | 0 | 0 | 39 | 6 | 6b | 0 | 0 | 0 | 0 | 0.388 |
| Japan | 0 | 1 | 45 | 22 | 2b | 0 | 0 | 0 | 0 | 0.480 |
Because the numbers of tandem repeats for MATR-13 and MATR-14 were rounded up in the study from Japan (e.g., 1.9 and 3.7 were reported as 2 and 4 copy numbers, respectively), the U.S. isolates were also rounded for those two loci, for comparison. These six loci had the greatest diversity among MATR loci studied among the isolates from Japan, but only three of the loci (MATR-2, MATR-3, and MATR-7) had comparable diversity among the U.S. isolates (9).
Copy number for M. avium subsp. hominissuis strain MAC 104.
DISCUSSION
We studied respiratory tract isolates and household water (biofilm) isolates from patients with nodular bronchiectatic MAC lung disease. Most isolates were recovered in the UTHSCT mycobacterial laboratory, and most patients lived within a 200-mile radius in northeast Texas. Isolates also included patient isolates and household water isolates from a Philadelphia suburb (Lankenau Medical Center) (20) and a small number of isolates from household water studies around the United States (5). We identified 49 VNTR types and eight subtypes (same VNTR number but identified with a or a b [e.g., 14a] to show a difference in the ITS sequence) of M. avium subsp. hominissuis based on the combination of the seven loci, including reference strains. Six VNTR types or subtypes were found only in an environmental source, 38 were identified only in a respiratory source, and 13 were identified in both sources. Eleven of 50 respiratory types (22%) contained isolates from three or more patients, while 33 types (66%) included only a single patient with that combination of seven VNTR patterns. The largest number of patients with a specific VNTR type, not associated with geographic clustering, was six patients.
The tandem-repeat loci in the current study were chosen from among 16 MATR-VNTR loci and four MIRU-VNTR loci (8, 9). We chose the MATR primers with the greatest allelic diversity (diversity values of ≥0.480) among respiratory isolates from Japan. It seems likely that some of the five larger current clusters (not clustered geographically) would have been separated into more VNTR types with the use of more loci (e.g., isolates from Japan are currently grouped using all 16 MATR loci) (9). For comparisons of isolates from the same patients or identification of geographic clusters, the current number of loci is likely adequate, less expensive, easier, and less time-consuming. For population studies, larger numbers of loci may be needed. Interestingly, all of the isolates in each of the larger clusters belonged to the same 3′ hsp65 code (sequevar). The six MATR loci chosen because of high allelic diversity in Japan were not as diverse among the U.S. isolates (especially MATR-1 and MATR-13) (Table 3), suggesting that an optimization study of different loci, similar to studies performed with Mycobacterium tuberculosis, would be needed for U.S. isolates of M. avium (17).
One of the VNTR types with multiple (four) patients (type 10) included reference strains ATCC 700898 (i.e., MAC 101) and MAC 104, both recovered in 1983 from the blood of AIDS patients (30). The isolates were indistinguishable by PFGE in the current study and were included in a study by Horan et al. that identified 10 patients, from five clinical sites in the western United States (30), with strains that were genotypically identical to MAC 104. Those authors utilized large sequence polymorphism typing as well as rep-PCR. That study supports the concept that some genotypes of M. avium are widespread geographically, as noted in the current study (30).
Tandem repeats rarely occur in exact multiples, and most VNTR loci contain complete as well as partial copies. Options for determining the numbers of tandem repeats include rounding up or down to a whole number or doing both, depending on the size of the partial copy (e.g., rounding down if the partial copy is <0.5 and rounding up if it is ≥0.5). We elected to report only the numbers of complete copies. This resulted in difficulties because with MATR-13 we encountered amplicons with 0.9 copies and 0 copies, both technically meeting the definition of 0 copies but differing in amplicon size. We elected to term the 0.9 copy as 0+ and the 0 copy (i.e., the amplicon includes only the flanking regions) as 0. This dilemma was not seen with any of the other loci. No consensus definition of copy numbers has been agreed upon for nontuberculous mycobacteria.
The majority of VNTR types had the Mav-B and Mav-A ITS sequences (which differ by a single base pair), which has been noted in other studies (1, 24, 28). For multiple isolates of the same VNTR type from the same patient, only one was sequenced; the results for the rest were presumed.
Three of 49 VNTR types (6.1%) of M. avium subsp. hominissuis, which included 13 patients, had the Mav-F ITS sequence. This sequevar of ITS was first noted in 2002 in a study by Mijs et al. of the M. avium complex in the Netherlands and, to our knowledge, has appeared only rarely in subsequent MAC studies in Europe and the United States (1, 2, 24). All three VNTR types with the Mav-F sequence exhibited tandem-repeat copy numbers for one or more loci seen infrequently among the other VNTR types (e.g., only 6 of the 52 VNTR types exhibited two tandem-repeat copies with MATR-7, including all VNTR types with the Mav-F ITS sequence). This was similar to the difference noted in a recent VNTR study of M. intracellulare in a single VNTR type that was found to be a different but closely related species (M. chimaera) (R. J. Wallace, Jr., unpublished observations) (12). The two species are also closely related in sequencing of the 16S rRNA gene (with a single base pair difference in the complete sequences) and the hsp65 gene but differ in the ITS and the rpoB gene (12, 31).
The unique character of M. avium VNTR types containing a Mav-F ITS sequence was underscored by the recognition that the isolates of these three VNTR types were all PCR positive using IS901 primers. Only 4 of 49 VNTR types (8.2%) and 0 of 8 subtypes were IS901 PCR positive. Upon sequencing, the insertion element was found to have 100% identity to ISMav6, which was first reported in 2009 by Ichikawa et al. for Japanese isolates of M. avium subsp. hominissuis and which has 95% sequence identity to IS901 (29). ISMav6 was found in 61% of 146 respiratory isolates, 87% of bathroom water isolates, and 0.0% of porcine isolates from Japan. No ITS sequencing of the isolates was performed (32). The importance of sequencing of the IS901 PCR amplicon was emphasized by a 2014 study by Tran and Han of 257 clinical strains of M. avium (33). Those authors found that 19/257 U.S. isolates (7.4%) of M. avium produced amplicons with IS901 primers, and they presumed the isolates were M. avium subsp. avium, without sequencing. We suspect those isolates carried ISMav6 and not IS901 (33).
The dramatic difference in the distributions of 3′ hsp65 sequevars (codes) (2), the distributions of different VNTR copy numbers for the same allelic site, and the low prevalence of ISMav6 in the southwestern and northeastern United States, compared to Japan, suggests that M. avium subsp. hominissuis isolates from different geographic areas may vary widely in their genetic composition. It also points out the potential for misleading findings if IS901 PCR results, without sequencing confirmation, are used for subspecies differentiation.
An interesting finding in the current study was that 8/13 (61.5%) of the multiple patients (five or more) with the same M. avium VNTR type were geographically clustered. Preliminary studies of one geographic area showed the same M. avium VNTR types in a local commercial water supplier and patient households (20).
There are limitations to this study. Single sputum samples with a unique VNTR type could not be validated by other methods. PFGE is so technically difficult, expensive, and time-consuming that its use for VNTR types with multiple isolates was limited (with the exception of support for the sequencing studies, this study was funded only by patient donations and institutional support). Thus, much of the testing resulted from clinical needs rather than testing of consecutive isolates. The use of PFGE for some isolates but not all might have resulted in a selection bias. Because VNTR analysis is so consistent, fast, and relatively inexpensive and does not require sequencing, it has replaced PFGE for comparisons of M. avium strains in our laboratory.
The creation of an international database is within reach for M. avium with the availability of VNTR analysis. The current VNTR method used the loci with the greatest allelic diversity and allowed for individual strain differentiation in the same patients and recognition of geographic clustering but was simple enough to be used clinically. The number of VNTR loci needed for optimal strain differentiation will need to be determined for larger population studies. The current technique does not require sequencing, with a few exceptions, but only PCR (six pairs or sets of isolates had the same VNTR copy numbers but differed in their ITS sequences). The finding that different patient isolates with the same VNTR type are mostly clonal by PFGE and 3′ hsp65 sequencing demonstrates that clonal groups do exist within M. avium. The presence of a typing system that identifies specific clones of M. avium should allow for comparisons with isolates from other diseases (e.g., disseminated disease or cavitary lung disease) and other geographic sources and thus the beginning of a risk analysis for M. avium in drinking water.
ACKNOWLEDGMENTS
This study was supported in part by institutional (UTHSCT) funds, MAC patient donors, and the Amon G. Carter Foundation.
We thank Joanne Woodring for excellent clerical assistance.
ADDENDUM IN PROOF
Since acceptance of this article, three new studies have been published describing M. avium subsp. hominissuis isolates harboring the ISMav6 element. A study by K. Ichikawa et al. (Infect Genet Evol 36:250–255, 2015, http://dx.doi.org/10.1016/j.meegid.2015.09.029) showed the high prevalence of ISMav6 from South Korea and Japan, but low percentages in select isolates from the Netherlands, Germany, and the United States. In C. Vluggen et al. (Euro Surveill. 21(3):pii=30111, 2016, http://dx.doi.org/10.2807/1560-7917.ES.2016.21.3.30111), two clinical isolates from Belgium were described as IS1245 negative and false IS901 positive (confirmed as ISMav6 by sequencing). In South Korea, S.-Y. Kim et al. (PLoS One 11(2):e0148917, 2016, http://dx.doi.org/10.1371/journal.pone.0148917) found that 61% of isolates contained the ISMav6 element (consistent with findings from Japan) and that these correlated with a higher resistance to moxifloxacin. ISMav6-positive isolates from all three studies harbored hsp65 codes 1, 2, 15, and 16.
REFERENCES
- 1.Mijs W, de Haas P, Rossau R, Van der Laan T, Rigouts L, Portaels F, van Soolingen D. 2002. Molecular evidence to support a proposal to reserve the designation Mycobacterium avium subsp. avium for bird-type isolates and ‘M avium subsp hominissuis’ for the human/porcine type of M avium. Int J Syst Evol Microbiol 52:1505–1518. doi: 10.1099/00207713-52-5-1505. [DOI] [PubMed] [Google Scholar]
- 2.Turenne CY, Semret M, Cousins DV, Collins DM, Behr MA. 2006. Sequencing of hsp65 distinguishes among subsets of the Mycobacterium avium complex. J Clin Microbiol 44:433–440. doi: 10.1128/JCM.44.2.433-440.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Wallace RJ Jr, Zhang Y, Brown BA, Dawson D, Murphy DT, Wilson R, Griffith DE. 1998. Polyclonal Mycobacterium avium complex infections in patients with nodular bronchiectasis. Am J Respir Crit Care Med 158:1235–1244. doi: 10.1164/ajrccm.158.4.9712098. [DOI] [PubMed] [Google Scholar]
- 4.Wallace RJ Jr, Zhang Y, Brown-Elliott BA, Yakrus MA, Wilson RW, Mann L, Couch L, Girard WM, Griffith DE. 2002. Repeat positive cultures in Mycobacterium intracellulare lung disease after macrolide therapy represent new infections in patients with nodular bronchiectasis. J Infect Dis 186:266–273. doi: 10.1086/341207. [DOI] [PubMed] [Google Scholar]
- 5.Falkinham JO., III 2011. Nontuberculous mycobacteria from household plumbing of patients with nontuberculous mycobacteria disease. Emerg Infect Dis 17:419–424. doi: 10.3201/eid1703.101510. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Zhang Y, Rajagopalan M, Brown BA, Wallace RJ Jr. 1997. Randomly amplified polymorphic DNA PCR for comparison of Mycobacterium abscessus strains from nosocomial outbreaks. J Clin Microbiol 35:3132–3139. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Li L, Bannantine JP, Zhang Q, Amonsin A, May BJ, Alt D, Banerji N, Kanjilal S, Kapur V. 2005. The complete genome sequence of Mycobacterium avium subspecies paratuberculosis. Proc Natl Acad Sci U S A 102:12344–12349. doi: 10.1073/pnas.0505662102. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Bull TJ, Sidi-Boumedine K, McMinn EJ, Stevenson K, Pickup R, Hermon-Taylor J. 2003. Mycobacterial interspersed repetitive units (MIRU) differentiate Mycobacterium avium subspecies paratuberculosis from other species of the Mycobacterium avium complex. Mol Cell Probes 17:157–164. doi: 10.1016/S0890-8508(03)00047-1. [DOI] [PubMed] [Google Scholar]
- 9.Inagaki T, Nishimori K, Yagi T, Ichikawa K, Moriyama M, Nakagawa T, Shibayama T, Uchiya K-I, Nikai T, Ogawa K. 2009. Comparison of a variable-number tandem-repeat (VNTR) method for typing Mycobacterium avium with mycobacterial interspersed repetitive-unit-VNTR and IS1245 restriction fragment length polymorphism typing. J Clin Microbiol 47:2156–2164. doi: 10.1128/JCM.02373-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Ablordey A, Swings J, Hubans C, Chemlal K, Locht C, Portaels F, Supply P. 2005. Multilocus variable-number tandem repeat typing of Mycobacterium ulcerans. J Clin Microbiol 43:1546–1551. doi: 10.1128/JCM.43.4.1546-1551.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Dauchy FA, Dégrange S, Charron A, Dupon M, Xin Y, Bébéar C, Maugein J. 2010. Variable-number tandem-repeat markers for typing Mycobacterium intracellulare strains isolated in humans. BMC Microbiol 10:93. doi: 10.1186/1471-2180-10-93. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Iakhiaeva E, McNulty S, Brown-Elliott BA, Falkinham JO III, Williams MD, Vasireddy R, Wilson RW, Turenne C, Wallace RJ Jr. 2013. Mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) genotyping of Mycobacterium intracellulare for strain comparison with establishment of a PCR database. J Clin Microbiol 51:409–416. doi: 10.1128/JCM.02443-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Ichikawa K, Yagi T, Inagaki T, Moriyama M, Nakagawa T, Uchiya KI, Nikal T, Ogawa K. 2010. Molecular type of Mycobacterium intracellulare using multilocus variable-number of tandem-repeat analysis: identification of loci and analysis of clinical isolates. Microbiology 156:496–504. doi: 10.1099/mic.0.030684-0. [DOI] [PubMed] [Google Scholar]
- 14.Martin A, Herranz M, Serrano MJ, Bouza E, Garcia de Viedma D. 2007. Rapid clonal analysis of recurrent tuberculosis by direct MIRU-VNTR typing on stored isolates. BMC Microbiol 7:73. doi: 10.1186/1471-2180-7-73. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Radomski N, Thibault VC, Karoui C, de Cruz K, Cochard T, Gutiérrez C, Supply P, Biet F, Boschiroli ML. 2010. Determination of genotypic diversity of Mycobacterium avium subspecies from human and animal origins by mycobacterial interspersed repetitive-unit-variable-number tandem-repeat and IS1311 restriction fragment length polymorphism typing methods. J Clin Microbiol 48:1026–1034. doi: 10.1128/JCM.01869-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Romano MI, Amadio A, Bigi F, Klepp L, Etchechoury I, Llana MN, Morsella C, Paolicchi F, Pavlik I, Bartos M, Leão SC, Cataldi A. 2005. Further analysis of VNTR and MIRU in the genome of Mycobacterium avium complex, and application to molecular epidemiology of isolates from South America. Vet Microbiol 110:221–237. doi: 10.1016/j.vetmic.2005.07.009. [DOI] [PubMed] [Google Scholar]
- 17.Supply P, Allix C, Lesjean S, Cardoso-Oelemann M, Rusch-Gerdes S, Willery E, Savine E, de Haas P, van Deutekom H, Roring S, Bifani P, Kurepina N, Kreiswirth B, Sola C, Rastogi N, Vatin V, Gutierrez MC, Fauville M, Niemann S, Skuce R, Kremer K, Locht C, van Soolingen D. 2006. Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variable-number tandem repeat typing of Mycobacterium tuberculosis. J Clin Microbiol 44:4498–4510. doi: 10.1128/JCM.01392-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Thibault VC, Grayon M, Boschiroli ML, Hibbans C, Overduin P, Stevenson K, Gutierrez MC, Supply P, Biet F. 2007. New variable-number tandem-repeat markers for typing Mycobacterium avium subsp. paratuberculosis and M. avium strains: comparison with IS900 and IS12456 restriction fragment length polymorphism typing. J Clin Microbiol 45:2404–2410. doi: 10.1128/JCM.00476-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Griffith DE, Aksamit T, Brown-Elliott BA, Catanzaro A, Daley C, Gordin F, Holland SM, Horsburgh R, Huitt G, Iademarco MF, Iseman M, Olivier K, Ruoss S, von Reyn CF, Wallace RJ Jr, Winthrop K. 2007. An official ATS/IDSA statement: diagnosis, treatment and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med 175:367–416. doi: 10.1164/rccm.200604-571ST. [DOI] [PubMed] [Google Scholar]
- 20.Lande L, Peterson DD, Sawicki J, Kwait R, Williams MD, Iakhiaeva E, Wallace RJ Jr, Falkinham JO III. 2013. Municipal water supply as a major source for pulmonary Mycobacterium avium pulmonary disease: a comparison of household and respiratory isolates. Am J Crit Care Med 187:A5100. [Google Scholar]
- 21.Wilton S, Cousins D. 1992. Detection and identification of multiple mycobacterial pathogens by DNA amplification in a single tube. PCR Methods Appl 1:269–273. doi: 10.1101/gr.1.4.269. [DOI] [PubMed] [Google Scholar]
- 22.Telenti A, Marchesi F, Balz M, Bally F, Böttger EC, Bodmer T. 1993. Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J Clin Microbiol 31:175–178. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Frothingham R, Wilson KH. 1994. Molecular phylogeny of the Mycobacterium avium complex demonstrates clinically meaningful divisions. J Infect Dis 169:305–312. doi: 10.1093/infdis/169.2.305. [DOI] [PubMed] [Google Scholar]
- 24.Stout JE, Hopkins GW, McDonald JR, Quinn A, Hamilton CD, Reller LB, Frothingham R. 2008. Association between 16S-23S internal transcribed spacer sequence groups of Mycobacterium avium complex and pulmonary disease. J Clin Microbiol 46:2790–2793. doi: 10.1128/JCM.00719-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, Swaminathan B. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 33:2233–2239. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Alvarez J, Gomez Garcia I, Aranaz A, Bozos J, Romero B, de Juan L, Mateos A, Gomez-Mampaso E, Domínguez L. 2008. Genetic diversity of Mycobacterium avium isolates recovered from clinical samples and from the environment: molecular characterization for diagnostic purposes. J Clin Microbiol 46:1246–1251. doi: 10.1128/JCM.01621-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Selander RK, Caugant DA, Ochman H, Musser JM, Gilmour MN, Whittam TS. 1986. Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics. Appl Environ Microbiol 51:873–884. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Frothingham R, Wilson KH. 1993. Sequence-based differentiation of strains in the Mycobacterium avium complex. J Bacteriol 175:2818–2825. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Ichikawa K, Yagi T, Moriyama M, Inagaki T, Nakagawa T, Uchiya KI, Nikai T, Ogawa K. 2009. Characterization of Mycobacterium avium clinical isolates in Japan using subspecies-specific insertion sequences, and identification of a new insertion sequence, ISMav6. J Med Microbiol 58:945–950. doi 10.1099/jmm.0.008623-0. [DOI] [PubMed] [Google Scholar]
- 30.Horan KL, Freeman R, Weigel K, Semret M, Pfaller S, Covert TC, van Soolingen D, Leao SC, Behr MA, Cangelosi GA. 2006. Isolation of the genome sequence strain Mycobacterium avium 104 from multiple patients over a 17-year period. J Clin Microbiol 44:783–789. doi: 10.1128/JCM.44.3.783-789.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Tortoli E, Rindi L, Garcia MJ, Chiaradonna P, Dei R, Garzelli C, Kroppenstedt RM, Lari N, Mattei R, Mariottini A, Mazzarelli G, Murcia MI, Nanetti A, Piccoli P, Scarparo C. 2004. Proposal to elevate the genetic variant MAC-A, included in the Mycobacterium avium complex, to species rank as Mycobacterium chimaera sp. nov. Int J Syst Evol Microbiol 54:1277–1285. doi: 10.1099/ijs.0.02777-0. [DOI] [PubMed] [Google Scholar]
- 32.Iwamoto T, Nakajima C, Nishiuchi Y, Kato T, Yoshida S, Nakanishi N, Tamaru A, Tamura Y, Suzuki Y, Nasu M. 2012. Genetic diversity of Mycobacterium avium subspecies hominissuis strains isolated from humans, pigs, and human living environment. Infect Genet Evol 12:846–852. doi: 10.1016/j.meegid.2011.06.018. [DOI] [PubMed] [Google Scholar]
- 33.Tran QT, Han XY. 2014. Subspecies identification and significance of 257 clinical strains of Mycobacterium avium. J Clin Microbiol 52:1201–1206. doi: 10.1128/JCM.03399-13. [DOI] [PMC free article] [PubMed] [Google Scholar]