JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1990, p. 2051-2058 0095-1137/90/092051-08$02.00/0 Copyright © 1990, American Society for Microbiology
Vol. 28, No. 9
Insertion Element IS986 from Mycobacterium tuberculosis: a Useful Tool for Diagnosis and Epidemiology of Tuberculosis PETER W. M. HERMANS,l* DICK
VAN SOOLINGEN,' JEREMY W. DALE,2 ANJA R. J. SCHUITEMA,3 RUTH A. McADAM,4t DAVID CATTY,4 AND JAN D. A. VAN EMBDEN1
National Institute of Public Health and Environmental Protection, P.O. Box 1, 3720 BA Bilthoven,' N. H. Swellengrebel Laboratory of Tropical Hygiene, Royal Tropical Institute, Amsterdam,3 The Netherlands, and Department of Microbiology, University of Surrey, Guildford, Surrey GU2 5XH,2 and Department of Immunology, The Medical School, University of Birmingham, Birmingham B15 2TJ,4 United Kingdom
and
Received 14 March 1990/Accepted 21 May 1990
IS986 of Mycobacterium tuberculosis belongs to the IS3-like family of insertion sequences, and it has previously been shown to be present in multiple copies in the chromosome of M. tuberculosis. In this study we investigated the value of a IS986-based DNA probe in the diagnosis and epidemiology of tuberculosis. IS986 was found only in species belonging to the M. tuberculosis complex. Independent isolates of M. tuberculosis complex strains showed a very high degree of polymorphism of restriction fragments which contained IS986 DNA. In contrast, Mycobacterium bovis BCG vaccine strains as well as clinical isolates of M. bovis BCG contained one copy of IS986, which was present at the same location in the chromosome. Different M. tuberculosis isolates from a recent M. tuberculosis outbreak showed an identical banding pattern. We concluded that IS986 is an extremely suitable tool for the diagnosis and epidemiology of tuberculosis. Tuberculosis is a highly contagious disease that is mainly transmitted from person to person. Because of the long incubation time needed for definitive laboratory diagnosis of this disease, the tracing of individuals who might have been in contact with infected people is a major strategy for limiting the dissemination of Mycobacterium tuberculosis. The typing of strains from infected individuals could play an important role in tracking the sources of infection. Phage typing (4) can be used for differentiating several groups of M. tuberculosis strains but is of limited value in the epidemiology of tuberculosis because only a few different phage types can be recognized (7). In addition, phage typing is a rather time-consuming and cumbersome technique which can be carried out by only a very specialized laboratory. Serotyping, on the other hand, cannot differentiate strains within the M. tuberculosis complex (10). Therefore, there is a great need for an improved method to subtype M. tuberculosis strains by a simple and rapid method. Restriction fragment length polymorphism (RFLP) in genomic DNA is commonly exploited to detect genetic variability (9). Several investigators have identified M. tuberculosis DNA elements which are present in multiple copies per genome and which show a polymorphic banding pattern among the few isolates that have been investigated (6, 15, 24). One of these elements, IS986, was recently sequenced and was found to share considerable homology with insertion sequences of the IS3 family of enterobacteria (R. A. McAdam, P. W. M. Hermans, D. van Soolingen, Z. F. Zainuddin, D. Catty, J. D. A. van Embden, and J. W. Dale, submitted for publication). The DNA sequence was virtually identical to the recently described M. tuberculosis insertion element IS6110 (20). Although the transposition of these elements has not yet been described in M. tuberculosis, the sequence data indi*
Corresponding author.
t Present address: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY 10461.
cate that the insertion element is a functional transposable element. The 1,358-kilobase-pair (kb) element is flanked by inverted repeats of 30 base pairs (bp), and it contains a large open reading frame which shares homology with the transposase of the enterobacterial IS3 family. In this study, we evaluated the usefulness of IS986 as a probe for RFLP analysis. For this purpose, we analyzed a large number of isolates of M. tuberculosis complex strains from both epidemiologically nonrelated and related sources.
MATERIALS AND METHODS Bacterial strains, genomic DNA, and plasmids. The bacterial strains and plasmids used in this study and their origins are listed in Table 1. Media, reagents, and enzymes were used as described previously (21). Culturing of the mycobacterial strains and isolation of genomic DNA was performed as reported by Hermans et al. (12a). Labeling of DNA probes. The mycobacterial DNA fragments of pRP5000 were labeled with [a-32P]dCTP by using the multiprime DNA labeling kit (Amersham International plc, Amersham, United Kingdom) or with horseradish peroxidase by using the enhanced chemiluminescence gene detection system (Amersham International plc). The detection of horseradish peroxidase-labeled probes was carried out by the peroxidase-catalyzed oxidation of luminol and subsequent enhanced chemiluminescence. The emitted light was detected on X-ray film (16, 23). Southern blot hybridization. Digests of chromosomal DNAs were electrophoretically separated on 0.8% agarose gels containing ethidium bromide (500 ng/ml). After denaturation and transfer to Gene Screen Plus membranes (Dupont, NEN Research Products, Boston, Mass.) by vacuum blotting (14, 18) (Milliblot V-system; Millipore Corp., Bedford, Mass.), the DNA was hybridized with 32P-labeled probes, as described by Noordhoek et al. (13). Hybridization and washing were done at 65°C. Membranes were exposed to X-Omat film (Eastman Kodak Co., Rochester, N.Y.) at -70°C for various lengths of time. The experimental proce2051
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TABLE 1. Bacterial strains and plasmids used in this study Property
Strain or plasmid
Species
Strains 13, 14, 15, 19, 21, 22, 23, 32 3, 5, 10 92, 93, 95-101 107-128, 130-133 38 40, 41 43, 45, 105, 106 102 103 104 46 50 53 56 60 65 67 71 90 129
M. tuberculosis M. tuberculosis M. tuberculosis M. tuberculosis M. africanum M. bovis M. bovis BCG M. bovis BCG M. bovis BCG M. bovis BCG M. microti M. avium M. kansasii M. intracellulare M. fortuitum M. scrofulaceum M. smegmatis M. gordonae M. leprae Unknown; atypical strain
Plasmids pAT153 pRP5000
or
or Source reference
origin
Clinical isolates Clinical isolates (Ghana) Clinical isolates (outbreak in The Netherlands) Clinical isolates (Tilburg, The Netherlands) Clinical isolate Clinical isolates Clinical isolates Vaccine strain Vaccine strain Vaccine strain
ATCCf
ATCC 10143
This laboratory KIT9 P. L. van Puttenb
Clinical isolate (Tilburg, The Netherlands) Ampicillin resistance; pBR322 derivative pAT153 derivative carrying a 4.6-kb insertion element-containing M. tuberculosis DNA fragment pEX2 recombinant containing a 2.4-kb DNA fragment of M. tuberculosis
pPH7301
This laboratory T. van der Werft This laboratory P. L. van Puttenb This laboratory This laboratory This laboratory Organon Teknikac Armand Frappierd This laboratory F. Portaelse This laboratory This laboratory This laboratory This laboratory This laboratory
22 R. A. McAdam
This laboratory
aSophia Hospital, Zwolle, The Netherlands. b St. Elisabeth Hospital, Tilburg, The Netherlands. C Organon Teknika N.V., Turnhout, Belgium. d Institut Armand Frappier, Laval, Quebec, Canada. e Prince Leopold Institute of Tropical Medicine, Antwerp, Belgium. f American Type Culture Collection, Rockville, Md. g N. H. Swellengrebel Laboratory of Tropical Hygiene, Royal Tropical Institute, Amsterdam, The Netherlands.
dures for the enhanced chemiluminescence gene detection system recommended by the manufacturer were used. Synthetic oligonucleotides. Based on the sequence of IS986 (McAdam et al., submitted), two oligonucleotides were synthesized by using a DNA synthesizer (Applied Biosystems, Inc., Foster City, Calif.). The oligonucleotides INS-1 (5'-CGTGAGGGCATCGAGGTGGC) and INS-2 (5'-GCG TAGGCGTCGGTGACAAA) correspond to bp 631 to 650 and 856 to 875 of the IS986 sequence, respectively. Oligonucleotides INS-1 and INS-2 were complementary to opposite strands and positioned 205 bp apart. Two homologous regions within the 16S rRNA genes of M. bovis BCG (19) and the 16S rRNA genes from various other procaryotic species described by Dams et al. (5) were selected and used for
synthesizing the oligonucleotides R (5'-GTGCCAGCAGC CGCGGTAA) and S (5'-GTGCGGGCCCCCGTCAATTC). These oligonucleotides correspond to residues 504 to 523 and 910 to 929 on the opposite strands of the M. bovis BCG gene, respectively. Polymerase chain reaction. The polymerase chain reaction (PCR) (17) was performed with thermoresistent DNA polymerase from Thermus aquaticus (Taq polymerase; The Perkin-Elmer Corp., Norwalk, Conn.) as recommended by the manufacturer. One unit of Taq polymerase was added to 100 ,ul of 50 mM NaCl-2 mM MgC12-10 mM Tris hydrochloride-0.01% (wt/vol) gelatin-0.2 mM of each of the deoxynucleotides dGTP, dATP, dTTP, and dCTP (Boehringer GmbH, Mannheim, Federal Republic of Germany) (pH 9.6),
XhoI
EcoRI
BspMII PvuII
BamHI
BspM I
PvuII
EcoRI
4-r IR
IR ORF
500bp (IR). Also, the large 1,037-bp repeats Physical map of the 4.6-kb EcoRI insert of pRP5000 containing IS986 flanked by inverted open reading frame (ORF) is depicted.
FIG. 1.
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FIG. 2. Southern blot analysis of PvuII-digested chromosomal DNA of various mycobacterial species hybridized with the 32P-labeled 4.6-kb EcoRI fragment of pRP5000 (A) and the 2.4-kb EcoRI fragment of pPH7301 (B). Lanes 1, M. leprae 90; lanes 2, M. tuberculosis 13; lanes 3, M. tuberculosis 14; lanes 4, M. tuberculosis 15; lanes 5, M. tuberculosis 19; lanes 6, M. tuberculosis 23; lanes 7, M. tuberculosis 21; lanes 8, M. tuberculosis 22; lanes 9, M. tuberculosis 32; lanes 10, M. tuberculosis 10; lanes 11, M. bovis 40; lanes 12, M. bovis 41; lanes 13, M. bovis BCG 45; lanes 14, M. africanum 38; lanes 15, M. microti 46; lanes 16, M. avium 50; lanes 17, M. fortuitum 60; lanes 18, M. gordonae 71; lanes 19, M. intracellulare 56; lanes 20, M. kansasii 53. Numbers on the left indicate sizes of standard DNA fragments in kilobase pairs.
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FIG. 3. Southern blot analysis of PvuIl-digested chromosomal DNA of various mycobacterial species with the 32P-labeled 386-bp BamHI-Xhol fragment of pRP5000 used as a probe. Lane 1, M. leprae 90; lane 2, M. tuberculosis 14; lane 3, M. tuberculosis 15; lane 4, M. tuberculosis 19; lane 5, M. tuberculosis 23; lane 6, M. tuberculosis 21; lane 7, M. tuberculosis 22; lane 8, M. tuberculosis 32; lane 9, M. tuberculosis 10; lane 10, M. bovis 40; lane 11, M. bovis 41; lane 12, M. bovis BCG 45; lane 13, M. africanum 38; lane 14, M. microti 46; lane 15, M. avium 50; lane 16, M. fortuitum 60; lane 17, M. gordonae 71; lane 18, M. intracellulare 56; lane 19, M. kansasii 53. Numbers on the left indicate sizes of standard DNA fragments in kilobase pairs.
containing 100 ng of target DNA and 500 ng of each primer. DNA amplification was performed in a PCR processor (Bio-med GmbH, Theres, Federal Republic of Germany) with 35 temperature cycles: 1 min, 94°C; 1 min, 65°C; and 2 min, 72°C. The amplified DNA was analyzed by agarose gel electrophoresis. Animal experiments. TRL guinea pigs (weight, about 250 g) were subcutaneously and intramuscularly inoculated in one leg with 0.5 ml of a M. tuberculosis suspension of 102 bacilli per 1 ml of phosphate-buffered saline. After 8 weeks, the animals were sacrificed, M. tuberculosis was isolated from the spleens, and the bacteria were further cultured in vitro. RESULTS Occurrence of IS986 among the various mycobacterial species. In order to investigate the occurrence of IS986 in mycobacteria, plasmid pRP5000 was used for the isolation of the insertion element. pRP5000 is a derivative of a lambda recombinant that was selected from a M. tuberculosis gene library as a result of its hybridization to the M. fortuitum plasmid pUS300 (24). The physical map of the mycobacterial insert of pRP5000 is shown in Fig. 1. The 4.6-kb EcoRI insert was isolated and hybridized with PvuII-digested genomic DNA from different mycobacterial species. Multiple hybridizing bands were observed in DNA from M. tuberculosis, M. africanum, M. bovis, M. bovis BCG, and M. microti, whereas the insert did not hybridize with strains of M. leprae, M. avium, M. fortuitum, M. gordonae, M. intracel-
lulare, M. kansasii (Fig. 2A), M. flavescens, M. malmoense, M. phlei, M. scrofulaceum, M. paratuberculosis, M. chelonei, M. smegmatis, or M. perigrinum (data not shown). These results indicate that the occurrence of the IS986-like elements is restricted to strains belonging to the M. tuberculosis complex group. Polymorphism of restriction fragments containing IS986. All 14 M. tuberculosis complex strains shown in Fig. 2A contained multiple DNA fragments which hybridized with the 4.6-kb insert of pRP5000, which is consistent with the previous findings of Zainuddin and Dale (24). Furthermore, each strain displayed a different, unique banding pattern. To demonstrate that this RFLP is a special property of the fragments containing the IS986 element, we rehybridized the same blot with the 2.4-kb EcoRI fragment of pPH7301. This plasmid contains another repeated sequence that is present in the M. tuberculosis complex species M. gordonae and M. kansasii (12a). In contrast to the hybridization patterns obtained with the 4.6-kb EcoRI fragment of pRP5000, virtually identical banding patterns were observed among the different M. tuberculosis complex strains (Fig. 2B). Sequence data showed that IS986 is located between two BspMII sites (McAdam et al., submitted) which are unique sites in the inverted repeats of this insertion sequence element (Fig. 1). To confirm that the RFLP is due to restriction fragments that contain IS986, we used the 386-bp BamHI-XhoI fragment located within the insertion element of pRP5000 as a probe for the hybridization of PvuIIdigested chromosomal DNA of the same mycobacterial
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FIG. 4. RFLP analysis of M. bovis BCG strains. M. tuberculosis 15 (lane 1), M. tuberculosis 10 (lane 2), M. bovis BCG 102 (lane 3), M. bovis BCG 103 (lane 4), M. bovis BCG 104 (lane 5), M. bovis BCG 45 (lane 6), M. bovis BCG 43 (lane 7), M. bovis BCG 105 (lane 8), and M. bovis BCG 106 (lane 9) were analyzed. Southern blotting was performed as described in the legend to Fig. 3. Numbers on the left indicate sizes of standard DNA fragments in kilobase pairs.
strains used in Fig. 2. As expected, this small fragment did not hybridize with species other than those of the M.
tuberculosis complex. The banding patterns obtained with the M. tuberculosis complex strains were similar but not identical to those obtained with the 4.6-kb EcoRI probe (Fig. 3). This is consistent with the idea that the RFLP is due to the presence of IS986 at different sites in the genome of the various strains that we analyzed. IS986 in M. bovis BCG strains. As shown in Fig. 3, lane 12, only one fragment in M. bovis BCG hybridized with the 386-bp IS986-specific DNA probe. This indicates that IS986 is present as a single copy in this clinical isolate. We examined six additional M. bovis BCG strains, both vaccine strains as well as clinical isolates. All M. bovis BCG strains tested were found to contain a unique 1.7-kb PvuII fragment that hybridized with the 386-bp fragment IS986 (Fig. 4). We conclude that IS986 is present as a single copy in all M. bovis BCG strains and that it is inserted at the same location in the genome.
Specific DNA amplification of IS986 by PCR. Based on the of IS986 (McAdam et al., submitted), two oligonucleotides, INS-1 and INS-2, were synthesized. These oligonucleotides were used as primers for amplification by PCR of chromosomal DNA corresponding to residues 641 to 885 of IS986. As expected, after amplification of either M. tuberculosis DNA or pRP5000, a fragment of approximately 245 bp was visible on agarose gels (Fig. 5A). Chromosomal DNAs of M. africanum, M. bovis, M. bovis BCG, and M. microti were also found to contain the 245-bp amplifiable fragment. Furthermore, chromosomal DNAs from 22 M. sequence
FIG. 5. Agarose gel electrophoresis of amplified DNAs of different mycobacterial strains by using oligonucleotide amplimer pairs INS-1 and INS-2 (A) and R and S (B) in PCR. Lanes 1, pRP5000; lanes 2, M. tuberculosis 13; lanes 3, M. africanum 38; lanes 4, M. bovis 40; lanes 5, M. bovis BCG 104; lanes 6, M. microti 46; lanes 7, M. avium 50; lanes 8, M. kansasii 53; lanes 9, M. fortuitum 60; lanes 10, M. scrofulaceum 65; lanes 11, M. smegmatis 67; lanes 12, M. leprae 90. Numbers on the left indicate sizes of standard DNA fragments in base pairs.
tuberculosis strains, 6 M. bovis BCG strains, and 1 M. bovis strain were tested by PCR; and all were shown to contain the 245-bp amplifiable fragment (data not shown). In contrast, no such fragment was found in M. avium, M. kansasii, M. fortuitum, M. scrofulaceum, M. smegmatis, or M. leprae. As a positive control for successful DNA amplification in vitro, the two oligonucleotide primers R and S, which are homologous to the conserved regions of the procaryotic 16S rRNA genes (5, 19), were used to test the genomic DNA isolates. This resulted in the amplification of a 426-bp fragment from all mycobacterial species mentioned above (Fig. 5B). Genetic stability of IS986 during animal passage. The high degree of RFLP in IS986-containing sequences of M. tuberculosis complex strains suggests that this insertion element could transpose with a high frequency. To investigate whether genetic rearrangement of IS986 occurs after passage in animals susceptible to M. tuberculosis, we analyzed the restriction fragment patterns of four strains before and after passage during 2 months in guinea pigs. The results are shown in Fig. 6. No changes in the banding pattern were
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observed, indicating that IS986 is stable during a limited number of generations in vivo. Use of IS986 for epidemiological purposes. The observed RFLP caused by IS986 suggests the possibility that IS986 can be used for precise epidemiological investigations. Therefore, we tested an additional 15 clinical isolates of M. tuberculosis. These isolates were obtained from various regional hospitals and laboratories in The Netherlands. A total of 6 of the 15 strains were not known to originate from epidemiologically related cases of tuberculosis, and these strains showed different PvuII banding patterns when the 386-bp IS986 probe was used (data not shown). This is in marked contrast to the remaining nine isolates, which exhibited a mutually identical restriction fragment pattern (Fig. 7, lanes 1 to 9). These nine isolates originated from an outbreak of tuberculosis among individuals who were all treated by the same physician, who specialized in the treatment of patients with arthritis. The identities of the banding patterns from these isolates from a known outbreak indicate the potential value of IS986 as a probe for such studies. Further confirmation of the usefulness of this probe was provided by a study of 26 M. tuberculosis isolates which were obtained from a single regional health laboratory in January 1990; at that time, these isolates were not known to be related. Twenty-three different banding patterns were found among these isolates (Fig. 8). The banding patterns of two strains (Fig. 8, lanes 1 and 14) were identical to those shown in Fig. 7. Investigation of the patients' histories revealed that they could be traced to the same outbreak. An additional three strains (Fig. 8, lanes 15, 24, and 25) also showed character-
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FIG. 7. RFLP analysis of nine strains from an M. tuberculosis outbreak (lanes 1 to 9) and two nonrelated strains (lanes 10 and 11). M. tuberculosis 92 (lane 1), M. tuberculosis 93 (lane 2), M. tuberculosis 95 (lane 3), M. tuberculosis 96 (lane 4), M. tuberculosis 97 (lane 5), M. tuberculosis 98 (lane 6), M. tuberculosis 99 (lane 7), M. tuberculosis 100 (lane 8), M. tuberculosis 101 (lane 9), M. tuberculosis 15 (lane 10), and M. tuberculosis 10 (lane 11) were analyzed. Southern blotting was carried out as described in the legend to Fig. 3. Numbers on the left indicate sizes of standard DNA fragments in kilobase pairs.
istic banding patterns which were not related to the patterns of the strains involved in the outbreak. Case histories of these patients revealed that they were suspected of being contacts of a common source of infection. The application of the probe in this case was therefore successful in revealing a previously unsuspected cluster of cases of tuberculosis.
DISCUSSION As repetitive segments of DNA are found in virtually all procaryotic and eucaryotic organisms (8), it is not surprising that these elements have recently also been identified in mycobacteria. Clark-Curtiss and colleagues (2, 3, 12) found a 2.2-kb sequence that was present in at least 19 copies per M. leprae genome but that was not present in other mycobacterial species. Another species-specific repetitive DNA element in M. paratuberculosis was identified by Green et al. (11). This repeated sequence has significant homology with ISJlO, an insertion element of Streptomyces coelicolor. Various strains of M. leprae and M. paratuberculosis have been analyzed for RFLP by using the repetitive DNA as a probe, and the nonvariability of the banding patterns indicated that these elements were inserted at a specific position in the Mycobacterium chromosome. In M. tuberculosis, repetitive DNA elements have been found that are inserted at a specific location in the genome (12a), whereas others were found to differ in genomic position from strain to strain (6, 15, 24). In this study, we exploited the latter property of
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FIG. 8. RFLP analysis of 26 M. tuberculosis isolates obtained from a regional health laboratory. M. tuberculosis strains 107 (lane 1), 108 (lane 2), 109 (lane 3), 110 (lane 4), 111 (lane 5), 112 (lane 6), 113 (lane 7), 114 (lane 8), 115 (lane 9), 116 (lane 10), 117 (lane 11), 118 (lane 12), 119 (lane 13), 120 (lane 14), 121 (lane 15), 122 (lane 16), 123 (lane 17), 124 (lane 18), 125 (lane 19), 126 (lane 20) and 127 (lane 21), 128 (lane 22), 130 (lane 24), 131 (lane 25), 132 (lane 26) and 133 (lane 27) and the unknown atypical mycobacterial strain 129 (lane 23) were analyzed. The Southern blot was hybridized with the horseradish peroxidase-labeled BamHI-XhoI fragment. Numbers on the left indicate sizes of standard DNA fragments in kilobase pairs.
the putative insertion sequence IS986. This element is 1,386 bp in size and is flanked by inverted repeats that are homologous to those of IS3 and IS3411. Furthermore, IS986 contains an open reading frame, which encodes a putative transposase, which is significantly homologous in its amino acid sequence to the transposases of the IS3 family of enterobacterial insertion elements (McAdam et al., submitted). A similar but not identical element was recently sequenced by Thierry et al. (20). In this study, we showed that IS986-like elements are present in 1 to 20 copies per genome and that these elements are specific for the species of the M. tuberculosis complex. We extend the preliminary observations of Zainuddin and Dale (24) that these elements differ in their chromosomal location from strain to strain. The patterns of the restriction fragments containing IS986 in all 32 independent isolates of M. tuberculosis strains were different. Such an extreme degree of polymorphism reinforces the idea that IS986 is a functional insertion element that can insert to new sites on the same replicon and that can initiate other types of genetic rearrangements such as cointegration of replicons, inversion, and deletion of sequences adjacent to the insertion sequence element (8). The latter type of genetic event has recently been suggested by Hermans et al. (12a) to be a consequence of the activity of another repetitive DNA element in M. tuberculosis. This study shows that the high degree of polymorphism of IS986-containing fragments is very useful as a tool in determining the epidemiology of tuberculosis. All strains from an epidemiologically welldefined outbreak of tuberculosis were of the same characteristic RFLP type. Furthermore, in a collection of 26 strains obtained from a peripheral laboratory in the southern part of The Netherlands, we could identify two strains with this RFLP pattern. Later, it appeared that these strains origi-
nated from patients involved in the same outbreak mentioned above. Also, three strains with another characteristic RFLP pattern were recognized, and these strains were suspected of being contacts with individuals involved in a single tuberculosis outbreak. We are in the process of determining the RFLP types of all M. tuberculosis strains that are sent to our reference laboratory. By computer analysis of a library of previously established RFLP patterns, one might be able to trace sources of M. tuberculosis from the distant past. An interesting observation was the nonpolymorphic pattern of IS986-containing restriction fragments of M. bovis BCG. This derivative was obtained in 1921 from a pathogenic Mycobacterium strain that lost its virulence gradually during passages in liquid medium (1). Since then, many substrains of M. bovis BCG have been used to prepare vaccines around the world, and these substrains are heterogeneous in many of their characteristics. As all strains investigated in this study were of the same RFLP type and contained a single copy of IS986 in their genomes, all M. bovis BCG isolates are likely to contain a single IS986 copy at one particular location in the chromosome. This suggests that IS986 lost its transposition capability before or during the 230 passages of the parental strain of M. bovis BCG and that all substrains are identical with respect to the site of insertion of IS986 in the chromosome. This unique location in all M. bovis BCG strains will be useful in distinguishing M. bovis BCG from other mycobacteria. We will investigate whether there is a correlation between the loss of virulence of M. bovis BCG and the location of IS986 in the chromosome. The high polymorphism of fragments containing IS986 among clinical isolates of M. tuberculosis complex strains suggests either that IS986 changes frequently in its position in the chromosome or that IS986 is relatively stable but that
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the clinical isolates examined in this study have a long history of separate lineage. Our observations that no polymorphism was observed among strains from single outbreaks and that serial passage in guinea pigs does not lead to substrains with IS986 at a different chromosomal location point toward the latter possibility and suggest that IS986 is a relatively stable element which transposes with a low frequency. Because IS986 is found only in M. tuberculosis complex strains, this element is a useful target for in vitro amplification by PCR for the rapid and specific detection of such bacteria in clinical specimens, without the need for culturing these slowly growing microorganisms. ACKNOWLEDGMENTS We thank T. van der Werf, P. L. van Putten, and F. Portaels for providing us with mycobacterial strains and J. G. Baas for helpful discussions and technical assistance. This study received financial support from the World Health Organization Programme for Vaccine Development. LITERATURE CITED 1. Calmette, A., L. Negre, and A. Boquet. 1922. Essais de vaccination du lapin et du cobaye contre l'infection tuberculeuse. Ann. Inst. Pasteur (Paris) 36:625-631. 2. Clark-Curtiss, J., and M. A. Docherty. 1989. A species-specific repetitive sequence in Mycobacterium leprae DNA. J. Infect. Dis. 159:7-15. 3. Clark-Curtiss, J., and G. P. Walsh. 1989. Conservation of genomic sequences among isolates of Mycobacterium tuberculosis. J. Bacteriol. 171:4844-4851. 4. Crawford, J., and J. H. Bates. 1984. Phage typing of mycobacteria, p. 123-132. In G. P. Kubica and L. G. Wayne (ed.), The mycobacteria: a sourcebook. Part A. Marcel Dekker, Inc., New York. 5. Dams, E., L. Hendriks, Y. Van de Peer, J. Neefs, G. Smits, I. Vandenbempt, and R. De Wachter. 1988. Compilation of small ribosomal RNA sequences. Nucleic Acids Res. 16:87-172. 6. Eisenach, K. D., J. T. Crawford, and J. H. Bates. 1988. Repetitive DNA sequence as probes for Mycobacterium tuberculosis. J. Clin. Microbiol. 26:2240-2245. 7. Engel, H. W. B. 1978. Mycobacteriophage and phage typing. Ann. Microbiol. 129A:75-90. 8. Galas, D. J., and M. Chandler. 1989. Bacterial insertion sequences, p. 109-162. In D. E. Berg and M. M. Howe (ed.), Mobile DNA. American Society for Microbiology, Washington,
D.C. 9. Gill, P., A. J. Jeffreys, and D. J. Werrett. 1985. Forensic application of DNA 'fingerprint.' Nature (London) 318:577-579. 10. Good, R. C., and R. E. Beam. 1984. Seroagglutination, p. 105-122. In G. P. Kubica and L. G. Wayne (ed.), The mycobacteria: a sourcebook. Part A. Marcel Dekker, Inc., New York. 11. Green, E. P., M. L. V. Tizard, M. T. Moss, J. Thompson, D. J. Winterbourne, J. J. McFadden, and J. Hermon-Taylor. 1989.
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Sequence and characteristics of IS900, an insertion element identified in a human Crohn's disease isolate of Mycobacterium paratuberculosis. Nucleic Acids Res. 17:9063-9073. 12. Grosskinsky, C. M., W. R. Jacobs, Jr., J. E. Clark-Curtiss, and B. R. Bloom. 1989. Genetic relationships among Mycobacterium leprae, Mycobacterium tuberculosis, and candidate leprosy vaccine strains determined by DNA hybridization: identification of an M. leprae-specific repetitive sequence. Infect. Immun. 57:1535-1541. 12a.Hermans, P. W. M., A. R. J. Schuitema, D. van Soolingen, C. P. H. J. Verstynen, E. M. Bik, J. E. R. Thole, A. H. J. Kolk, and J. D. A. van Emden. 1990. Specific detection of Mycobacterium tuberculosis complex strains by polymerase chain reaction. J. Clin. Microbiol. 28:1204-1213. 13. Noordhoek, G. T., P. W. M. Hermans, L. M. Schouls, J. J. van der Sluis, and J. D. A. van Embden. 1989. Treponema pallidum subspecies pallidum (Nichols) and Treponema pallidum subspecies pertenue differ in at least one nucleotide: comparison of two homologous antigens. Microb. Pathog. 6:29-42. 14. Olsewska, E., and K. Jones. 1988. Vacuum blotting enhances nucleic acid transfer. Trends Genet. 4:92-94. 15. Reddi, P. P., G. P. Talwar, and P. S. Khandekar. 1988. Repetitive DNA sequence of Mycobacterium tuberculosis: analysis of differential hybridization pattern with other mycobacteria. Int. J. Lepr. 56:592-597. 16. Renz, M., and C. Kurz. 1984. A colorimetric method for DNA hybridization. Nucleic Acids Res. 12:3435-3444. 17. Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, G. T. Horn, K. B. Mullis, and H. A. Erlich. 1988. Primerdirected enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-491. 18. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 97:503-517. 19. Suzuki, Y., A. Nagata, Y. Ono, and T. Yamada. 1988. Complete nucleotide sequence of the 16S rRNA gene of Mycobacterium bovis BCG. J. Bacteriol. 170:2886-2889. 20. Thierry, D., M. D. Cave, K. D. Eisenach, J. T. Crawford, J. H. Bates, B. Gicquel, and J. L. Guesdon. 1990. IS6110, an IS-like element of Mycobacterium tuberculosis complex. Nucleic Acids Res. 18:188. 21. Thole, J. E. R., W. C. Keulen, A. H. J. Kolk, D. G. Groothuis, L. G. Berwald, R. H. Tiesjema, and J. D. A. van Embden. 1987. Characterization, sequence determination, and immunogenicity of a 64-kilodalton protein of Mycobacterium bovis BCG expressed in Escherichia coli. Infect. Immun. 55:1466-1475. 22. Twigg, A. J., and D. Sherratt. 1980. Trans-complementable copy-number mutants of plasmid ColEL. Nature (London) 283: 216-218. 23. Whitehead, T. P., G. H. G. Thorpe, T. J. N. Carter, and L. J. Kricka. 1983. Enhanced luminescence procedure for sensitive determination of peroxidase labelled conjugates in immunoassay. Nature (London) 305:158-159. 24. Zainuddin, Z. F., and J. W. Dale. 1989. Polymorphic repetitive DNA sequences in Mycobacterium tuberculosis detected with a gene probe from a Mycobacterium fortuitum plasmid. J. Gen. Microbiol. 135:2347-2355.
Vol. 28, No. 9
Insertion Element IS986 from Mycobacterium tuberculosis: a Useful Tool for Diagnosis and Epidemiology of Tuberculosis PETER W. M. HERMANS,l* DICK
VAN SOOLINGEN,' JEREMY W. DALE,2 ANJA R. J. SCHUITEMA,3 RUTH A. McADAM,4t DAVID CATTY,4 AND JAN D. A. VAN EMBDEN1
National Institute of Public Health and Environmental Protection, P.O. Box 1, 3720 BA Bilthoven,' N. H. Swellengrebel Laboratory of Tropical Hygiene, Royal Tropical Institute, Amsterdam,3 The Netherlands, and Department of Microbiology, University of Surrey, Guildford, Surrey GU2 5XH,2 and Department of Immunology, The Medical School, University of Birmingham, Birmingham B15 2TJ,4 United Kingdom
and
Received 14 March 1990/Accepted 21 May 1990
IS986 of Mycobacterium tuberculosis belongs to the IS3-like family of insertion sequences, and it has previously been shown to be present in multiple copies in the chromosome of M. tuberculosis. In this study we investigated the value of a IS986-based DNA probe in the diagnosis and epidemiology of tuberculosis. IS986 was found only in species belonging to the M. tuberculosis complex. Independent isolates of M. tuberculosis complex strains showed a very high degree of polymorphism of restriction fragments which contained IS986 DNA. In contrast, Mycobacterium bovis BCG vaccine strains as well as clinical isolates of M. bovis BCG contained one copy of IS986, which was present at the same location in the chromosome. Different M. tuberculosis isolates from a recent M. tuberculosis outbreak showed an identical banding pattern. We concluded that IS986 is an extremely suitable tool for the diagnosis and epidemiology of tuberculosis. Tuberculosis is a highly contagious disease that is mainly transmitted from person to person. Because of the long incubation time needed for definitive laboratory diagnosis of this disease, the tracing of individuals who might have been in contact with infected people is a major strategy for limiting the dissemination of Mycobacterium tuberculosis. The typing of strains from infected individuals could play an important role in tracking the sources of infection. Phage typing (4) can be used for differentiating several groups of M. tuberculosis strains but is of limited value in the epidemiology of tuberculosis because only a few different phage types can be recognized (7). In addition, phage typing is a rather time-consuming and cumbersome technique which can be carried out by only a very specialized laboratory. Serotyping, on the other hand, cannot differentiate strains within the M. tuberculosis complex (10). Therefore, there is a great need for an improved method to subtype M. tuberculosis strains by a simple and rapid method. Restriction fragment length polymorphism (RFLP) in genomic DNA is commonly exploited to detect genetic variability (9). Several investigators have identified M. tuberculosis DNA elements which are present in multiple copies per genome and which show a polymorphic banding pattern among the few isolates that have been investigated (6, 15, 24). One of these elements, IS986, was recently sequenced and was found to share considerable homology with insertion sequences of the IS3 family of enterobacteria (R. A. McAdam, P. W. M. Hermans, D. van Soolingen, Z. F. Zainuddin, D. Catty, J. D. A. van Embden, and J. W. Dale, submitted for publication). The DNA sequence was virtually identical to the recently described M. tuberculosis insertion element IS6110 (20). Although the transposition of these elements has not yet been described in M. tuberculosis, the sequence data indi*
Corresponding author.
t Present address: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY 10461.
cate that the insertion element is a functional transposable element. The 1,358-kilobase-pair (kb) element is flanked by inverted repeats of 30 base pairs (bp), and it contains a large open reading frame which shares homology with the transposase of the enterobacterial IS3 family. In this study, we evaluated the usefulness of IS986 as a probe for RFLP analysis. For this purpose, we analyzed a large number of isolates of M. tuberculosis complex strains from both epidemiologically nonrelated and related sources.
MATERIALS AND METHODS Bacterial strains, genomic DNA, and plasmids. The bacterial strains and plasmids used in this study and their origins are listed in Table 1. Media, reagents, and enzymes were used as described previously (21). Culturing of the mycobacterial strains and isolation of genomic DNA was performed as reported by Hermans et al. (12a). Labeling of DNA probes. The mycobacterial DNA fragments of pRP5000 were labeled with [a-32P]dCTP by using the multiprime DNA labeling kit (Amersham International plc, Amersham, United Kingdom) or with horseradish peroxidase by using the enhanced chemiluminescence gene detection system (Amersham International plc). The detection of horseradish peroxidase-labeled probes was carried out by the peroxidase-catalyzed oxidation of luminol and subsequent enhanced chemiluminescence. The emitted light was detected on X-ray film (16, 23). Southern blot hybridization. Digests of chromosomal DNAs were electrophoretically separated on 0.8% agarose gels containing ethidium bromide (500 ng/ml). After denaturation and transfer to Gene Screen Plus membranes (Dupont, NEN Research Products, Boston, Mass.) by vacuum blotting (14, 18) (Milliblot V-system; Millipore Corp., Bedford, Mass.), the DNA was hybridized with 32P-labeled probes, as described by Noordhoek et al. (13). Hybridization and washing were done at 65°C. Membranes were exposed to X-Omat film (Eastman Kodak Co., Rochester, N.Y.) at -70°C for various lengths of time. The experimental proce2051
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TABLE 1. Bacterial strains and plasmids used in this study Property
Strain or plasmid
Species
Strains 13, 14, 15, 19, 21, 22, 23, 32 3, 5, 10 92, 93, 95-101 107-128, 130-133 38 40, 41 43, 45, 105, 106 102 103 104 46 50 53 56 60 65 67 71 90 129
M. tuberculosis M. tuberculosis M. tuberculosis M. tuberculosis M. africanum M. bovis M. bovis BCG M. bovis BCG M. bovis BCG M. bovis BCG M. microti M. avium M. kansasii M. intracellulare M. fortuitum M. scrofulaceum M. smegmatis M. gordonae M. leprae Unknown; atypical strain
Plasmids pAT153 pRP5000
or
or Source reference
origin
Clinical isolates Clinical isolates (Ghana) Clinical isolates (outbreak in The Netherlands) Clinical isolates (Tilburg, The Netherlands) Clinical isolate Clinical isolates Clinical isolates Vaccine strain Vaccine strain Vaccine strain
ATCCf
ATCC 10143
This laboratory KIT9 P. L. van Puttenb
Clinical isolate (Tilburg, The Netherlands) Ampicillin resistance; pBR322 derivative pAT153 derivative carrying a 4.6-kb insertion element-containing M. tuberculosis DNA fragment pEX2 recombinant containing a 2.4-kb DNA fragment of M. tuberculosis
pPH7301
This laboratory T. van der Werft This laboratory P. L. van Puttenb This laboratory This laboratory This laboratory Organon Teknikac Armand Frappierd This laboratory F. Portaelse This laboratory This laboratory This laboratory This laboratory This laboratory
22 R. A. McAdam
This laboratory
aSophia Hospital, Zwolle, The Netherlands. b St. Elisabeth Hospital, Tilburg, The Netherlands. C Organon Teknika N.V., Turnhout, Belgium. d Institut Armand Frappier, Laval, Quebec, Canada. e Prince Leopold Institute of Tropical Medicine, Antwerp, Belgium. f American Type Culture Collection, Rockville, Md. g N. H. Swellengrebel Laboratory of Tropical Hygiene, Royal Tropical Institute, Amsterdam, The Netherlands.
dures for the enhanced chemiluminescence gene detection system recommended by the manufacturer were used. Synthetic oligonucleotides. Based on the sequence of IS986 (McAdam et al., submitted), two oligonucleotides were synthesized by using a DNA synthesizer (Applied Biosystems, Inc., Foster City, Calif.). The oligonucleotides INS-1 (5'-CGTGAGGGCATCGAGGTGGC) and INS-2 (5'-GCG TAGGCGTCGGTGACAAA) correspond to bp 631 to 650 and 856 to 875 of the IS986 sequence, respectively. Oligonucleotides INS-1 and INS-2 were complementary to opposite strands and positioned 205 bp apart. Two homologous regions within the 16S rRNA genes of M. bovis BCG (19) and the 16S rRNA genes from various other procaryotic species described by Dams et al. (5) were selected and used for
synthesizing the oligonucleotides R (5'-GTGCCAGCAGC CGCGGTAA) and S (5'-GTGCGGGCCCCCGTCAATTC). These oligonucleotides correspond to residues 504 to 523 and 910 to 929 on the opposite strands of the M. bovis BCG gene, respectively. Polymerase chain reaction. The polymerase chain reaction (PCR) (17) was performed with thermoresistent DNA polymerase from Thermus aquaticus (Taq polymerase; The Perkin-Elmer Corp., Norwalk, Conn.) as recommended by the manufacturer. One unit of Taq polymerase was added to 100 ,ul of 50 mM NaCl-2 mM MgC12-10 mM Tris hydrochloride-0.01% (wt/vol) gelatin-0.2 mM of each of the deoxynucleotides dGTP, dATP, dTTP, and dCTP (Boehringer GmbH, Mannheim, Federal Republic of Germany) (pH 9.6),
XhoI
EcoRI
BspMII PvuII
BamHI
BspM I
PvuII
EcoRI
4-r IR
IR ORF
500bp (IR). Also, the large 1,037-bp repeats Physical map of the 4.6-kb EcoRI insert of pRP5000 containing IS986 flanked by inverted open reading frame (ORF) is depicted.
FIG. 1.
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FIG. 2. Southern blot analysis of PvuII-digested chromosomal DNA of various mycobacterial species hybridized with the 32P-labeled 4.6-kb EcoRI fragment of pRP5000 (A) and the 2.4-kb EcoRI fragment of pPH7301 (B). Lanes 1, M. leprae 90; lanes 2, M. tuberculosis 13; lanes 3, M. tuberculosis 14; lanes 4, M. tuberculosis 15; lanes 5, M. tuberculosis 19; lanes 6, M. tuberculosis 23; lanes 7, M. tuberculosis 21; lanes 8, M. tuberculosis 22; lanes 9, M. tuberculosis 32; lanes 10, M. tuberculosis 10; lanes 11, M. bovis 40; lanes 12, M. bovis 41; lanes 13, M. bovis BCG 45; lanes 14, M. africanum 38; lanes 15, M. microti 46; lanes 16, M. avium 50; lanes 17, M. fortuitum 60; lanes 18, M. gordonae 71; lanes 19, M. intracellulare 56; lanes 20, M. kansasii 53. Numbers on the left indicate sizes of standard DNA fragments in kilobase pairs.
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FIG. 3. Southern blot analysis of PvuIl-digested chromosomal DNA of various mycobacterial species with the 32P-labeled 386-bp BamHI-Xhol fragment of pRP5000 used as a probe. Lane 1, M. leprae 90; lane 2, M. tuberculosis 14; lane 3, M. tuberculosis 15; lane 4, M. tuberculosis 19; lane 5, M. tuberculosis 23; lane 6, M. tuberculosis 21; lane 7, M. tuberculosis 22; lane 8, M. tuberculosis 32; lane 9, M. tuberculosis 10; lane 10, M. bovis 40; lane 11, M. bovis 41; lane 12, M. bovis BCG 45; lane 13, M. africanum 38; lane 14, M. microti 46; lane 15, M. avium 50; lane 16, M. fortuitum 60; lane 17, M. gordonae 71; lane 18, M. intracellulare 56; lane 19, M. kansasii 53. Numbers on the left indicate sizes of standard DNA fragments in kilobase pairs.
containing 100 ng of target DNA and 500 ng of each primer. DNA amplification was performed in a PCR processor (Bio-med GmbH, Theres, Federal Republic of Germany) with 35 temperature cycles: 1 min, 94°C; 1 min, 65°C; and 2 min, 72°C. The amplified DNA was analyzed by agarose gel electrophoresis. Animal experiments. TRL guinea pigs (weight, about 250 g) were subcutaneously and intramuscularly inoculated in one leg with 0.5 ml of a M. tuberculosis suspension of 102 bacilli per 1 ml of phosphate-buffered saline. After 8 weeks, the animals were sacrificed, M. tuberculosis was isolated from the spleens, and the bacteria were further cultured in vitro. RESULTS Occurrence of IS986 among the various mycobacterial species. In order to investigate the occurrence of IS986 in mycobacteria, plasmid pRP5000 was used for the isolation of the insertion element. pRP5000 is a derivative of a lambda recombinant that was selected from a M. tuberculosis gene library as a result of its hybridization to the M. fortuitum plasmid pUS300 (24). The physical map of the mycobacterial insert of pRP5000 is shown in Fig. 1. The 4.6-kb EcoRI insert was isolated and hybridized with PvuII-digested genomic DNA from different mycobacterial species. Multiple hybridizing bands were observed in DNA from M. tuberculosis, M. africanum, M. bovis, M. bovis BCG, and M. microti, whereas the insert did not hybridize with strains of M. leprae, M. avium, M. fortuitum, M. gordonae, M. intracel-
lulare, M. kansasii (Fig. 2A), M. flavescens, M. malmoense, M. phlei, M. scrofulaceum, M. paratuberculosis, M. chelonei, M. smegmatis, or M. perigrinum (data not shown). These results indicate that the occurrence of the IS986-like elements is restricted to strains belonging to the M. tuberculosis complex group. Polymorphism of restriction fragments containing IS986. All 14 M. tuberculosis complex strains shown in Fig. 2A contained multiple DNA fragments which hybridized with the 4.6-kb insert of pRP5000, which is consistent with the previous findings of Zainuddin and Dale (24). Furthermore, each strain displayed a different, unique banding pattern. To demonstrate that this RFLP is a special property of the fragments containing the IS986 element, we rehybridized the same blot with the 2.4-kb EcoRI fragment of pPH7301. This plasmid contains another repeated sequence that is present in the M. tuberculosis complex species M. gordonae and M. kansasii (12a). In contrast to the hybridization patterns obtained with the 4.6-kb EcoRI fragment of pRP5000, virtually identical banding patterns were observed among the different M. tuberculosis complex strains (Fig. 2B). Sequence data showed that IS986 is located between two BspMII sites (McAdam et al., submitted) which are unique sites in the inverted repeats of this insertion sequence element (Fig. 1). To confirm that the RFLP is due to restriction fragments that contain IS986, we used the 386-bp BamHI-XhoI fragment located within the insertion element of pRP5000 as a probe for the hybridization of PvuIIdigested chromosomal DNA of the same mycobacterial
VOL. 28, 1990
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FIG. 4. RFLP analysis of M. bovis BCG strains. M. tuberculosis 15 (lane 1), M. tuberculosis 10 (lane 2), M. bovis BCG 102 (lane 3), M. bovis BCG 103 (lane 4), M. bovis BCG 104 (lane 5), M. bovis BCG 45 (lane 6), M. bovis BCG 43 (lane 7), M. bovis BCG 105 (lane 8), and M. bovis BCG 106 (lane 9) were analyzed. Southern blotting was performed as described in the legend to Fig. 3. Numbers on the left indicate sizes of standard DNA fragments in kilobase pairs.
strains used in Fig. 2. As expected, this small fragment did not hybridize with species other than those of the M.
tuberculosis complex. The banding patterns obtained with the M. tuberculosis complex strains were similar but not identical to those obtained with the 4.6-kb EcoRI probe (Fig. 3). This is consistent with the idea that the RFLP is due to the presence of IS986 at different sites in the genome of the various strains that we analyzed. IS986 in M. bovis BCG strains. As shown in Fig. 3, lane 12, only one fragment in M. bovis BCG hybridized with the 386-bp IS986-specific DNA probe. This indicates that IS986 is present as a single copy in this clinical isolate. We examined six additional M. bovis BCG strains, both vaccine strains as well as clinical isolates. All M. bovis BCG strains tested were found to contain a unique 1.7-kb PvuII fragment that hybridized with the 386-bp fragment IS986 (Fig. 4). We conclude that IS986 is present as a single copy in all M. bovis BCG strains and that it is inserted at the same location in the genome.
Specific DNA amplification of IS986 by PCR. Based on the of IS986 (McAdam et al., submitted), two oligonucleotides, INS-1 and INS-2, were synthesized. These oligonucleotides were used as primers for amplification by PCR of chromosomal DNA corresponding to residues 641 to 885 of IS986. As expected, after amplification of either M. tuberculosis DNA or pRP5000, a fragment of approximately 245 bp was visible on agarose gels (Fig. 5A). Chromosomal DNAs of M. africanum, M. bovis, M. bovis BCG, and M. microti were also found to contain the 245-bp amplifiable fragment. Furthermore, chromosomal DNAs from 22 M. sequence
FIG. 5. Agarose gel electrophoresis of amplified DNAs of different mycobacterial strains by using oligonucleotide amplimer pairs INS-1 and INS-2 (A) and R and S (B) in PCR. Lanes 1, pRP5000; lanes 2, M. tuberculosis 13; lanes 3, M. africanum 38; lanes 4, M. bovis 40; lanes 5, M. bovis BCG 104; lanes 6, M. microti 46; lanes 7, M. avium 50; lanes 8, M. kansasii 53; lanes 9, M. fortuitum 60; lanes 10, M. scrofulaceum 65; lanes 11, M. smegmatis 67; lanes 12, M. leprae 90. Numbers on the left indicate sizes of standard DNA fragments in base pairs.
tuberculosis strains, 6 M. bovis BCG strains, and 1 M. bovis strain were tested by PCR; and all were shown to contain the 245-bp amplifiable fragment (data not shown). In contrast, no such fragment was found in M. avium, M. kansasii, M. fortuitum, M. scrofulaceum, M. smegmatis, or M. leprae. As a positive control for successful DNA amplification in vitro, the two oligonucleotide primers R and S, which are homologous to the conserved regions of the procaryotic 16S rRNA genes (5, 19), were used to test the genomic DNA isolates. This resulted in the amplification of a 426-bp fragment from all mycobacterial species mentioned above (Fig. 5B). Genetic stability of IS986 during animal passage. The high degree of RFLP in IS986-containing sequences of M. tuberculosis complex strains suggests that this insertion element could transpose with a high frequency. To investigate whether genetic rearrangement of IS986 occurs after passage in animals susceptible to M. tuberculosis, we analyzed the restriction fragment patterns of four strains before and after passage during 2 months in guinea pigs. The results are shown in Fig. 6. No changes in the banding pattern were
2056
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observed, indicating that IS986 is stable during a limited number of generations in vivo. Use of IS986 for epidemiological purposes. The observed RFLP caused by IS986 suggests the possibility that IS986 can be used for precise epidemiological investigations. Therefore, we tested an additional 15 clinical isolates of M. tuberculosis. These isolates were obtained from various regional hospitals and laboratories in The Netherlands. A total of 6 of the 15 strains were not known to originate from epidemiologically related cases of tuberculosis, and these strains showed different PvuII banding patterns when the 386-bp IS986 probe was used (data not shown). This is in marked contrast to the remaining nine isolates, which exhibited a mutually identical restriction fragment pattern (Fig. 7, lanes 1 to 9). These nine isolates originated from an outbreak of tuberculosis among individuals who were all treated by the same physician, who specialized in the treatment of patients with arthritis. The identities of the banding patterns from these isolates from a known outbreak indicate the potential value of IS986 as a probe for such studies. Further confirmation of the usefulness of this probe was provided by a study of 26 M. tuberculosis isolates which were obtained from a single regional health laboratory in January 1990; at that time, these isolates were not known to be related. Twenty-three different banding patterns were found among these isolates (Fig. 8). The banding patterns of two strains (Fig. 8, lanes 1 and 14) were identical to those shown in Fig. 7. Investigation of the patients' histories revealed that they could be traced to the same outbreak. An additional three strains (Fig. 8, lanes 15, 24, and 25) also showed character-
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istic banding patterns which were not related to the patterns of the strains involved in the outbreak. Case histories of these patients revealed that they were suspected of being contacts of a common source of infection. The application of the probe in this case was therefore successful in revealing a previously unsuspected cluster of cases of tuberculosis.
DISCUSSION As repetitive segments of DNA are found in virtually all procaryotic and eucaryotic organisms (8), it is not surprising that these elements have recently also been identified in mycobacteria. Clark-Curtiss and colleagues (2, 3, 12) found a 2.2-kb sequence that was present in at least 19 copies per M. leprae genome but that was not present in other mycobacterial species. Another species-specific repetitive DNA element in M. paratuberculosis was identified by Green et al. (11). This repeated sequence has significant homology with ISJlO, an insertion element of Streptomyces coelicolor. Various strains of M. leprae and M. paratuberculosis have been analyzed for RFLP by using the repetitive DNA as a probe, and the nonvariability of the banding patterns indicated that these elements were inserted at a specific position in the Mycobacterium chromosome. In M. tuberculosis, repetitive DNA elements have been found that are inserted at a specific location in the genome (12a), whereas others were found to differ in genomic position from strain to strain (6, 15, 24). In this study, we exploited the latter property of
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FIG. 8. RFLP analysis of 26 M. tuberculosis isolates obtained from a regional health laboratory. M. tuberculosis strains 107 (lane 1), 108 (lane 2), 109 (lane 3), 110 (lane 4), 111 (lane 5), 112 (lane 6), 113 (lane 7), 114 (lane 8), 115 (lane 9), 116 (lane 10), 117 (lane 11), 118 (lane 12), 119 (lane 13), 120 (lane 14), 121 (lane 15), 122 (lane 16), 123 (lane 17), 124 (lane 18), 125 (lane 19), 126 (lane 20) and 127 (lane 21), 128 (lane 22), 130 (lane 24), 131 (lane 25), 132 (lane 26) and 133 (lane 27) and the unknown atypical mycobacterial strain 129 (lane 23) were analyzed. The Southern blot was hybridized with the horseradish peroxidase-labeled BamHI-XhoI fragment. Numbers on the left indicate sizes of standard DNA fragments in kilobase pairs.
the putative insertion sequence IS986. This element is 1,386 bp in size and is flanked by inverted repeats that are homologous to those of IS3 and IS3411. Furthermore, IS986 contains an open reading frame, which encodes a putative transposase, which is significantly homologous in its amino acid sequence to the transposases of the IS3 family of enterobacterial insertion elements (McAdam et al., submitted). A similar but not identical element was recently sequenced by Thierry et al. (20). In this study, we showed that IS986-like elements are present in 1 to 20 copies per genome and that these elements are specific for the species of the M. tuberculosis complex. We extend the preliminary observations of Zainuddin and Dale (24) that these elements differ in their chromosomal location from strain to strain. The patterns of the restriction fragments containing IS986 in all 32 independent isolates of M. tuberculosis strains were different. Such an extreme degree of polymorphism reinforces the idea that IS986 is a functional insertion element that can insert to new sites on the same replicon and that can initiate other types of genetic rearrangements such as cointegration of replicons, inversion, and deletion of sequences adjacent to the insertion sequence element (8). The latter type of genetic event has recently been suggested by Hermans et al. (12a) to be a consequence of the activity of another repetitive DNA element in M. tuberculosis. This study shows that the high degree of polymorphism of IS986-containing fragments is very useful as a tool in determining the epidemiology of tuberculosis. All strains from an epidemiologically welldefined outbreak of tuberculosis were of the same characteristic RFLP type. Furthermore, in a collection of 26 strains obtained from a peripheral laboratory in the southern part of The Netherlands, we could identify two strains with this RFLP pattern. Later, it appeared that these strains origi-
nated from patients involved in the same outbreak mentioned above. Also, three strains with another characteristic RFLP pattern were recognized, and these strains were suspected of being contacts with individuals involved in a single tuberculosis outbreak. We are in the process of determining the RFLP types of all M. tuberculosis strains that are sent to our reference laboratory. By computer analysis of a library of previously established RFLP patterns, one might be able to trace sources of M. tuberculosis from the distant past. An interesting observation was the nonpolymorphic pattern of IS986-containing restriction fragments of M. bovis BCG. This derivative was obtained in 1921 from a pathogenic Mycobacterium strain that lost its virulence gradually during passages in liquid medium (1). Since then, many substrains of M. bovis BCG have been used to prepare vaccines around the world, and these substrains are heterogeneous in many of their characteristics. As all strains investigated in this study were of the same RFLP type and contained a single copy of IS986 in their genomes, all M. bovis BCG isolates are likely to contain a single IS986 copy at one particular location in the chromosome. This suggests that IS986 lost its transposition capability before or during the 230 passages of the parental strain of M. bovis BCG and that all substrains are identical with respect to the site of insertion of IS986 in the chromosome. This unique location in all M. bovis BCG strains will be useful in distinguishing M. bovis BCG from other mycobacteria. We will investigate whether there is a correlation between the loss of virulence of M. bovis BCG and the location of IS986 in the chromosome. The high polymorphism of fragments containing IS986 among clinical isolates of M. tuberculosis complex strains suggests either that IS986 changes frequently in its position in the chromosome or that IS986 is relatively stable but that
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the clinical isolates examined in this study have a long history of separate lineage. Our observations that no polymorphism was observed among strains from single outbreaks and that serial passage in guinea pigs does not lead to substrains with IS986 at a different chromosomal location point toward the latter possibility and suggest that IS986 is a relatively stable element which transposes with a low frequency. Because IS986 is found only in M. tuberculosis complex strains, this element is a useful target for in vitro amplification by PCR for the rapid and specific detection of such bacteria in clinical specimens, without the need for culturing these slowly growing microorganisms. ACKNOWLEDGMENTS We thank T. van der Werf, P. L. van Putten, and F. Portaels for providing us with mycobacterial strains and J. G. Baas for helpful discussions and technical assistance. This study received financial support from the World Health Organization Programme for Vaccine Development. LITERATURE CITED 1. Calmette, A., L. Negre, and A. Boquet. 1922. Essais de vaccination du lapin et du cobaye contre l'infection tuberculeuse. Ann. Inst. Pasteur (Paris) 36:625-631. 2. Clark-Curtiss, J., and M. A. Docherty. 1989. A species-specific repetitive sequence in Mycobacterium leprae DNA. J. Infect. Dis. 159:7-15. 3. Clark-Curtiss, J., and G. P. Walsh. 1989. Conservation of genomic sequences among isolates of Mycobacterium tuberculosis. J. Bacteriol. 171:4844-4851. 4. Crawford, J., and J. H. Bates. 1984. Phage typing of mycobacteria, p. 123-132. In G. P. Kubica and L. G. Wayne (ed.), The mycobacteria: a sourcebook. Part A. Marcel Dekker, Inc., New York. 5. Dams, E., L. Hendriks, Y. Van de Peer, J. Neefs, G. Smits, I. Vandenbempt, and R. De Wachter. 1988. Compilation of small ribosomal RNA sequences. Nucleic Acids Res. 16:87-172. 6. Eisenach, K. D., J. T. Crawford, and J. H. Bates. 1988. Repetitive DNA sequence as probes for Mycobacterium tuberculosis. J. Clin. Microbiol. 26:2240-2245. 7. Engel, H. W. B. 1978. Mycobacteriophage and phage typing. Ann. Microbiol. 129A:75-90. 8. Galas, D. J., and M. Chandler. 1989. Bacterial insertion sequences, p. 109-162. In D. E. Berg and M. M. Howe (ed.), Mobile DNA. American Society for Microbiology, Washington,
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