GERALD H. MAZUREK,1* SANDRA HARTMAN,2 YANSHENG ZHANG,2 BARBARA A. BROWN,2. JANEL S. R. HECTOR,2 DAVID MURPHY,3 AND RICHARD J. WALLACE, JR. ..... Arbeit, R. D., M. Arthur, R. Dunn, C. Kim, R. K. Selander, and.
JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1993, P. 390-394
Vol. 31, No. 2
0095-1137/93/020390-05$02.00/0 Copyright © 1993, American Society for Microbiology
Large DNA Restriction Fragment Polymorphism in the Mycobactenium avium-M. intracellulare Complex: a Potential Epidemiologic Tool GERALD H. MAZUREK,1* SANDRA HARTMAN,2 YANSHENG ZHANG,2 BARBARA A. BROWN,2 JANEL S. R. HECTOR,2 DAVID MURPHY,3 AND RICHARD J. WALLACE, JR.2 Departments ofMedicine,1 Microbiology,2 and Pathology,3 The University of Texas Health Center at Tyler, P.O. Box 2003, Tyler, Texas 75710 Received 6 July 1992/Accepted 5 November 1992
Mycobacterium avium-M. intraceUldare complex (MAI) isolates were studied by comparing the large restriction fragment (LRF) patterns produced by digesting their DNAs with infrequently cutting restriction endonucleases and separating the resultant large fragments by pulsed-field gel electrophoresis. Four reference strains and 35 randomly selected clinical MAI isolates gave highly diverse LRF patterns when their DNAs were digested with XbaI or AsnI. The LRF patterns of random isolates identified to be the same species by DNA probe analysis were not similar. The LRF patterns of random isolates of the same serotype were also different. In contrast, all isolates recovered from the same patient gave identical patterns. This included 28 isolates from nine patients. One isolate from sputum, one isolate from bone marrow, and two isolates from blood recovered over a 27-month period from a patient with AIDS were identical. Seven isolates recovered from the sputum of a second patient over 37 months also had identical patterns. The LRF patterns of unrelated MAI strains are highly polymorphic, appear to be strain specific, are relatively stable, and offer exciting promise as epidemiologic markers for the study of MAI infections. Mycobacterium avium-M. intracellulare complex (MAI) refers to a heterogeneous collection of acid-fast organisms. These organisms can be isolated from numerous environmental sites including soil, water, and house dust (6, 10, 15, 20). They are associated with animal and human diseases including infections of the lung, lymph nodes, skin, bones, and gastrointestinal and genitourinary tracts (12, 13, 26). Disseminated human infections are common in people with AIDS (3, 7). Despite the prevalence of MAT infections, the epidemiology remains obscure. This is due in part to a lack of adequate strain-specific epidemiologic markers. Determination of growth requirements, antibiotic and heavy metal susceptibilities, serotype, bacteriophage type, plasmid profile, multilocus enzyme electrophoretic type, and species by DNA probe analysis has provided limited insight into MAI infections (5, 6, 8, 14, 17, 27, 28). Conventional restriction endonuclease analysis of bacterial chromosomal DNA has been used to assess the genetic relatedness of MAI members but has not allowed strain-specific identification because of the large number of restriction fragments produced and the complexity of the patterns generated (24, 25). The number of restriction fragments and the complexities of the digestion patterns can be reduced by using infrequently cutting restriction endonucleases. These enzymes, which have, on average, fewer than 10 recognition site per 106 bp, cut the chromosomal DNA into a few large fragments. The resultant large restriction fragments (LRFs) cannot be separated by conventional agarose electrophoresis but can be separated by pulsed-field gel electrophoresis (PFGE). PFGE can cleanly separate DNA fragments as large as 2 Mb (4). LRF patterns, which are produced by digesting genomic DNA with infrequently cutting restriction endonucleases and separating the resultant large fragments by PFGE, have been *
used to study numerous bacterial species (1, 2, 9, 18, 21, 26). Levy-Frebault et al. used infrequently cutting endonucleases and field inversion gel electrophoresis (a prototype of PFGE) to evaluate DNA polymorphism in M. paratuberculosis (16). We previously reported the use of LRF patterns for comparing sporadic and clustered (epidemic) isolates of M. fortuitum and M. tuberculosis (11, 29). In the study described here we created and compared the LRF patterns of 4 MAI reference strains, 35 randomly selected clinical MAI isolates, and nine clusters of related MAI isolates.
MATERIALS AND METHODS
Organisms. Sixty-three clinical MAI isolates were obtained from the Nocardia/Mycobacteria Research Laboratory of the University of Texas Health Center at Tyler. The isolates were submitted from the clinical laboratory of the University of Texas Health Center at Tyler or from other clinical laboratories for antibiotic susceptibility testing. Some isolates were previously serotyped in the laboratory of Anna Tsang at the National Jewish Hospital, Denver, Colo. (5 isolates), or in the Mycobacteriology Laboratory of the Centers for Disease Control, Atlanta, Ga. (18 isolates). Thirty-five isolates were selected randomly. Twenty-three isolates were selected because they were recovered from one of nine patients, as described in Table 1. Patient 1 provided seven isolates over a 37-month period, with three isolates recovered from separate specimens on a single day. Four individual colonies (531.5a through 531.5d, Table 1) of one isolate were studied in addition to the original culture. Isolates were collected on consecutive days from three patients (patients 2, 3, and 9). Patients 4, 5, and 8 provided isolates from multiple sites. Two patients (patients 6 and 7) provided sputum isolates 11 or more months apart. Isolates which were identified to the species level by serotype and/or
Corresponding author. 390
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5
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436.5339.5291.0242.5194.0145.597.048.5-
8
9
10
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12
13
14
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15
-436.5 -339.5 -291.0 -242.5 -194.0
--145.5 -97.0 -48.5
FIG. 1. XbaI LRF patterns of unrelated MAI strains. Lane 3, M. avium ATCC 35718; lane 4, M. intracellulare ATCC 35761; lane 5, M. intracellulare ATCC 35847; lane 2 and lanes 6 through 14, randomly selected clinical MAI isolates; lanes 1 and 15, lambda DNA concatemers.
DNA probe (Snap Probe; Digene Diagnostics Inc, Silver Springs, Md.) analysis were passaged no more than twice before being frozen at -70°C in tryptic soy broth with 15% glycerol until needed for study. Two reference M. avium strains, ATCC 35712 and ATCC 35718 (serotypes 2 and 3, respectively), and two reference M. intracellulare strains, ATCC 35761 and ATCC 35847 (serotypes 7 and 14, respectively), were obtained from the American Type Culture Collection, Rockville, Md., and were held frozen at -70°C in tryptic soy broth with 15% glycerol until needed for study. Liberation of intact mycobacterial DNA. Isolates were revived from frozen stocks on 7H10 plates supplemented with 10% OAD (0.06% saponified oleic acid, 5% bovine albumin fraction V, and 2% glucose). Multiple (sweeps) or single colonies were transferred to 20 ml of 7H9 broth supplemented with 10% OAD and 0.4% Tween 80, where they were grown at 37°C on a rotary shaker at 140 rpm to the late exponential phase (5 to 10 days). Cycloserine (1 mg/ml) and ampicillin (0.1 mg/ml) were added, and the cultures were incubated for an additional 24 h prior to harvesting. DNA was prepared as described by Levy-Frebault et al. (16), with modification. Briefly, cells were resuspended in 2 ml of 1% low-melting-point agarose, and the mixture was poured into plug molds. The plugs were incubated in a solution containing lysozyme (2 mg of lysozyme and 1 pl of 2-mercaptoethanol per ml of TE [10 mM Tris, 1 mM EDTA]) at 37°C for 2 h. The plugs were then incubated in a solution containing proteinase K (1 mg of proteinase K per ml of 1% sodium dodecyl sulfate in TE) for 48 h at 50°C. They were washed in phenylmethylsulfonyl fluoride solution (1 mM phenylmethylsulfonyl fluoride in TE) once and in TE three times for 30 min at 4°C and were then stored at 4°C until needed. Restriction endonuclease digestion and PFGE. Plugs containing DNA were digested overnight with 20 U of XbaI or AsnI in buffer as recommended by the manufacturer. Some strains were also digested with DraI, SspI, SpeI, and NdeI. Plugs containing digested DNA were loaded into a 1% agarose gel prepared and run in Tris-borate-EDTA buffer (0.025 M Tris, 0.5 mM EDTA, 0.025 M boric acid). PFGE was carried out with a CHEF-DR II system (Bio-Rad Laboratories, Richmond, Calif.) or CHEF-Mapper at 14WC for 20 to 24 h at 200 V, with ramped pulse times varied according to the enzyme used. Gels were stained with ethidium bromide
and photographed by using UV illumination. Molecular weight standards were included with each electrophoresis. The molecular weight standards were polymerized bacteriophage lambda DNA (Bio-Rad) and Saccharomyces cerevisiae (yeast) chromosomal DNA (Bio-Rad). RESULTS Each MAI isolate gave a readily discernible LRF pattern when its genomic DNA was digested with XbaI or AsnI and subjected to PFGE. Some strains were also digested with Dral, SspI, SpeI, and NdeI, but AsnI and XbaI gave clear patterns more consistently than the other enzymes studied. AsnI digestion produced fragments as large as 800 kb, with most being greater than 50 kb. XbaI digestion produced fragments of as large as 400 kb, with numerous fragments being less than 50 kb. DraI produced fragments of the largest size, up to 850 kb. SspI produced fragments as large as 300 kb. SpeI produced fragments as large as 700 kb. By adjusting the PFGE parameters for each enzyme, 15 to 20 of the largest fragments could be separated to produce discernible patterns. When DNAs from 4 reference strains and 35 randomly selected clinical MAI isolates were digested with XbaI, 39 different LRF patterns were recognized. The XbaI LRF patterns from some of these strains are shown in Fig. 1. The 15 unrelated strains known to be serotype 4 had markedly different LRF patterns, as did 4 serotype 8 strains. There were no detectable similarities in the LRF patterns of isolates which were identified to a particular species by serotype or DNA probe analysis. There were no detectable similarities in the LRF patterns of 15 randomly selected strains recovered from individuals residing in Houston, Tex., or 5 randomly isolated strains from Tulsa, Okla. AsnI digestion also produced 39 different patterns (data not shown). All MAI isolates recovered from any single patient gave identical LRF patterns (Table 1). The LRF patterns of the MAI isolates recovered from patient 1 were identical (Fig. 2, lanes 2 through 12). Additionally, four individual colonies of isolate 531.5 were compared, and each had an LRF pattern which was identical to that of the original culture (Fig. 2, lanes 8 through 11). Isolates recovered from patient 2 on 3 consecutive days had identical patterns, as did the same
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TABLE 1. Characteristics of isolates examined in the present study Patient no.
1 1 1 1 1 1 1 1 1 1 1
Culture date
patteIRa
Figure no.
Sputum Sputum Sputum Sputum Sputum Sputum Sputum Sputum Sputum Sputum Sputum
November 1989 25 February 1990 2 April 1991 2 April 1991 2 April 1991 7 August 1991 7 August 1991 7 August 1991 7 August 1991 7 August 1991 16 December 1991
x531 x531 x531 x531 x531 x531 x531 x531 x531 x531 x531
2/2 2/3 2/4 2/5 2/6 2/7 2/8 2/9 2/10 2/11 2/12
Isnolate
Culture site no.pat
531.0 531.1 531.2 531.3 531.4 531.5 531.5a 531.5b 531.5c 531.5d 531.6
lnno
2 2 2
5004.1 5004.2 5004.3
Sputum Sputum Sputum
16 September 1991 17 September 1991 18 September 1991
x5004 x5004 x5004
NSb
3 3 3
5001.1 5001.2 5001.3
Sputum Sputum Sputum
12 September 1991 13 September 1991 14 September 1991
xS001 xS001 x5001
NS NS NS
4 4 4 4
104.1 104.2 104.3 104.4
Bone marrow Stool Blood Blood
1 August 1985 8 July 1986 June 1987 3 November 1987
x104 x104 x 104 x 104
3/2 3/3 3/4 3/5
5 5
98.1 98.2
Lymph node Bone marrow
June 1985 June 1985
x98 x98
4/2 4/3
NS NS
6 6
513 684
Sputum Sputum
April 1990 March 1991
x513 x513
4/4 4/5
7 7
435.1 435.2
Sputum Sputum
3 August 1989 10 September 1990
x435 x435
4/6 4/7
8 8 8
608.1 608.2 608.3
Blood Stool Stool
15 August 1990 13 August 1990 29 April 1991
x608 x608 x608
4/8 4/9 4/10
9 9
64.1 64.2
Sputum Sputum
20 April 1984 20 April 1984
x64 x64
4/11 4/12
a
b
x indicates LRF pattern obtained using XbaI. NS, not shown.
2
number of isolates taken from patient 3 (data not shown). MAI was found in cultures of samples from multiple sites of patient 4 over a 27-month period. The four isolates had identical LRF patterns (Fig. 3, lanes 2 through 5). Two isolates from patient 5 were identical (Fig. 4, lanes 2 and 3), as were two isolates from patient 6 (Fig. 4, lanes 4 and 5), two isolates from patient 7 (Fig. 4, lanes 6 and 7), three isolates from patient 8 (Fig. 4, lanes 8 through 10), and two isolates from patient 9 (Fig. 4, lanes 11 and 12). AsnI digestion of these isolates produced similar clusters of LRF patterns (data not shown). DISCUSSION The results of the present study show that strains of MAT can be distinguished by using LRF patterns as strain-specific markers. Each of the 48 unrelated strains described (35 randomly selected isolates, 4 reference strains, and 9 clustered-isolate strains) gave a distinct LRF pattern with easily discernible fragment bands. Each strain could be identified and distinguished by its LRF pattern. No other reported method is as precise.
3
4 5
6
7
8
9
10
11
12
Kb 436.5-
291.0-
.?242 2. 5 194.0--
48,5-
FIG. 2. Xbal LRF patterns of MAI isolates from patient 1. Lanes 2 through 7, isolates from sputum recovered from November 1989 through August 1991 (isolates 531.0 through 531.5); lanes 8 through 11, single colonies from isolate 531.5 (531.5a through 531.5d); lane 12, sputum isolate recovered in December 1991 (531.6); lane 1, lambda DNA concatemers.
MMA LARGE RESTRICTION FRAGMENT PATTERNS
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Kb 436.5339.5 291.0 242.5194.0-
145.5 97.0-
48.5-
FIG. 3. XbaI LRF patterns of MAI isolates from patient 4. Lane 2, bone marrow isolate recovered in August 1985 (isolate 104.1); lane 3, stool isolate recovered in July 1986 (isolate 104.2); lane 4, blood isolate recovered in June 1987 (isolate 104.3); lane 5, blood isolate recovered in November 1987 (isolate 104.4); lane 1, lambda DNA concatemers.
Biochemical tests can differentiate members of MAI from other mycobacterial species. DNA probes can differentiate M. avium strains from M. intracellulare strains, with some exceptions (22). Seroagglutination allows separation of most MAI strains into 31 serotypes (23), but none of these techniques allow strain-specific identification. In contrast, LRF patterns appear to be strain specific, with a marked heterogeneity in the patterns produced by unrelated strains and similarity (identity) in the patterns produced by multiple isolates from the same patient.
1
2
3
4
Pt8
Pt7
Pt 6
Pt5
5
6
7
8
9
Pt9 10 11
12
436.5--339.5-
291.0 242.5194.0 145.597.0-
48.5-
FIG. 4. XbaI LRF patterns of MAI isolates from five unrelated patients. Lanes 2 and 3, isolates 98.1 and 98.2, respectively, from patient 5; lanes 4 and 5, isolates 513 and 684, respectively, from patient 6; lanes 6 and 7, isolates 435.1 and 435.2, respectively, from patient 7; lanes 8 through 10, isolates 608.1, 608.2, and 608.3, respectively, from patient 8; lanes 11 and 12, isolates 64.1 and 64.2 from patient 9; lane 1, lambda DNA concatemers.
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Frequently cutting restriction endonucleases and conventional agarose electrophoresis have been used to study MAI strains. Wards et al. (24) discovered substantial genetic heterogeneity when strains from 28 different MAI serotypes were digested with BstEII, PvuII, and Bcll. However, 18 of 20 serotype 2 isolates had identical patterns and 8 serotype 8 strains gave three patterns. For some strains the serotype could be predicted on the basis of the "restriction fragment length" type, but strain-specific identification was not possible. Picken et al. (19) separated 41 strains (serotypes 1 through 20) into eight groups on the basis of the restriction fragment length polymorphisms created with BamHI digestion, conventional agarose electrophoresis, and hybridization with a "mycobacterial common probe" (19). The strains were categorized to the species level on the basis of restriction fragment length patterns, but strain-specific identification was, again, not possible. Unlike Picken et al. (19) and Wards et al. (24), we were unable to identify similarities in the LRF patterns of unrelated strains of any particular serotype. When the LRF patterns of serotype 4 strains were compared, they were markedly different. Similarly, serotype 8 strains from unrelated patients could not be grouped on the basis of their LRF patterns. We were also unable to categorize strains to the species level on the basis of their LRF patterns. The LRF patterns were, however, strain specific. The LRF patterns for each patient appeared to be stable and reproducible over time. Individual colonies of one isolate were found to have identical LRF patterns. Multiple isolates recovered from the same individual on the same day and on consecutive days had identical LRF patterns. Even when isolates were recovered years apart their LRF patterns were identical. Seven isolates recovered over 37 months from one patient with chronic respiratory disease (patient 1) gave identical LRF patterns. Although not seen with the isolates examined in the present study, one or two LRF band differences have been seen in the LRF patterns of other mycobacteria that were epidemiologically related and presumed to be clonal (11, 29). LRF patterns provide a reliable and practical method of identifying a particular strain of MAI. The strain of MAI infecting patient 4 was identified by its distinctive LRF pattern. All four isolates recovered from patient 4 were identical, even though they were recovered from different sites. In a similar manner, the strain recovered from the bone marrow and lymph node of patient 5 could be identified and distinguished from the strain infecting the blood and stool of patient 8. Additional comparisons of isolates from patients with AIDS may identify portals of infection. Comparison of clinical and environmental isolates may identify sources of MAI infection. LRF patterns provide a reliable and practical method for comparing specific strains of MAI without the need for radioactivity or DNA probes. As such, LRF patterns produced by digesting genomic DNA with infrequently cutting endonucleases and separating the LRFs by PFGE will aid in the epidemiologic study of infections caused by MAI organisms. ACKNOWLEDGMENT This study was funded by a grant from the Lizanell & Colbert Coldwell Foundation. REFERENCES 1. Allardet-Servent, A., N. Bouziges, M. J. Carles-Nurit, G. Bourg, A. Gouby, and M. Ramuz. 1989. Use of low-frequency-cleavage
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