JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1990, p. 2051-20580095-1137/90/092051-08$02.00/0Copyright © 1990, American Society for Microbiology

Insertion Element IS986 from Mycobacterium tuberculosis:a Useful Tool for Diagnosis and Epidemiology of TuberculosisPETER 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,' and

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 ofImmunology, The Medical School,

University ofBirmingham, Birmingham B15 2TJ,4 United Kingdom

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 wasfound only in species belonging to the M. tuberculosis complex. Independent isolates ofM. tuberculosis complexstrains showed a very high degree of polymorphism of restriction fragments which contained IS986 DNA. Incontrast, 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 isolatesfrom 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 mainlytransmitted from person to person. Because of the longincubation time needed for definitive laboratory diagnosis ofthis disease, the tracing of individuals who might have beenin contact with infected people is a major strategy forlimiting the dissemination of Mycobacterium tuberculosis.The typing of strains from infected individuals could play an

important role in tracking the sources of infection. Phagetyping (4) can be used for differentiating several groups ofM.tuberculosis strains but is of limited value in the epidemiol-ogy of tuberculosis because only a few different phage typescan be recognized (7). In addition, phage typing is a rathertime-consuming and cumbersome technique which can becarried out by only a very specialized laboratory. Serotyp-ing, on the other hand, cannot differentiate strains within theM. tuberculosis complex (10). Therefore, there is a greatneed for an improved method to subtype M. tuberculosisstrains by a simple and rapid method. Restriction fragmentlength polymorphism (RFLP) in genomic DNA is commonlyexploited to detect genetic variability (9). Several investiga-tors have identified M. tuberculosis DNA elements whichare present in multiple copies per genome and which show a

polymorphic banding pattern among the few isolates thathave been investigated (6, 15, 24). One of these elements,IS986, was recently sequenced and was found to shareconsiderable homology with insertion sequences of the IS3family of enterobacteria (R. A. McAdam, P. W. M. Her-

mans, 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 recentlydescribed M. tuberculosis insertion element IS6110 (20).Although the transposition of these elements has not yetbeen 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 transposableelement. The 1,358-kilobase-pair (kb) element is flanked byinverted repeats of 30 base pairs (bp), and it contains a largeopen reading frame which shares homology with the trans-posase of the enterobacterial IS3 family. In this study, we

evaluated the usefulness of IS986 as a probe for RFLPanalysis. For this purpose, we analyzed a large number ofisolates of M. tuberculosis complex strains from both epide-miologically nonrelated and related sources.

MATERIALS AND METHODSBacterial strains, genomic DNA, and plasmids. The bacte-

rial strains and plasmids used in this study and their originsare listed in Table 1. Media, reagents, and enzymes were

used as described previously (21). Culturing of the mycobac-terial strains and isolation of genomic DNA was performedas reported by Hermans et al. (12a).

Labeling of DNA probes. The mycobacterial DNA frag-ments of pRP5000 were labeled with [a-32P]dCTP by usingthe multiprime DNA labeling kit (Amersham Internationalplc, Amersham, United Kingdom) or with horseradish per-oxidase by using the enhanced chemiluminescence genedetection system (Amersham International plc). The detec-tion of horseradish peroxidase-labeled probes was carriedout by the peroxidase-catalyzed oxidation of luminol and

subsequent enhanced chemiluminescence. The emitted lightwas detected on X-ray film (16, 23).

Southern blot hybridization. Digests of chromosomalDNAs were electrophoretically separated on 0.8% agarosegels containing ethidium bromide (500 ng/ml). After denatur-ation and transfer to Gene Screen Plus membranes (Dupont,NEN Research Products, Boston, Mass.) by vacuum blot-

ting (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 proce-

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J. CLIN. MICROBIOL.2052 HERMANS ET AL.

TABLE 1. Bacterial strains and plasmids used in this studySource or

Strain or plasmid Species Property or origin reference

Strains13, 14, 15, 19, 21, 22, 23, 32 M. tuberculosis Clinical isolates This laboratory3, 5, 10 M. tuberculosis Clinical isolates (Ghana) T. van der Werft92, 93, 95-101 M. tuberculosis Clinical isolates (outbreak in The Netherlands) This laboratory107-128, 130-133 M. tuberculosis Clinical isolates (Tilburg, The Netherlands) P. L. van Puttenb38 M. africanum Clinical isolate This laboratory40, 41 M. bovis Clinical isolates This laboratory43, 45, 105, 106 M. bovis BCG Clinical isolates This laboratory102 M. bovis BCG Vaccine strain Organon Teknikac103 M. bovis BCG Vaccine strain Armand Frappierd104 M. bovis BCG Vaccine strain This laboratory46 M. microti F. Portaelse50 M. avium This laboratory53 M. kansasii This laboratory56 M. intracellulare This laboratory60 M. fortuitum This laboratory65 M. scrofulaceum This laboratory67 M. smegmatis ATCC 10143 ATCCf71 M. gordonae This laboratory90 M. leprae KIT9129 Unknown; atypical strain Clinical isolate (Tilburg, The Netherlands) P. L. van Puttenb

PlasmidspAT153 Ampicillin resistance; pBR322 derivative 22pRP5000 pAT153 derivative carrying a 4.6-kb insertion R. A. McAdam

element-containing M. tuberculosis DNAfragment

pPH7301 pEX2 recombinant containing a 2.4-kb DNA This laboratoryfragment of M. tuberculosis

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 detectionsystem recommended by the manufacturer were used.

Synthetic oligonucleotides. Based on the sequence of IS986(McAdam et al., submitted), two oligonucleotides weresynthesized by using a DNA synthesizer (Applied Biosys-tems, Inc., Foster City, Calif.). The oligonucleotides INS-1(5'-CGTGAGGGCATCGAGGTGGC) and INS-2 (5'-GCGTAGGCGTCGGTGACAAA) correspond to bp 631 to 650and 856 to 875 of the IS986 sequence, respectively. Oligo-nucleotides INS-1 and INS-2 were complementary to oppo-site strands and positioned 205 bp apart. Two homologousregions within the 16S rRNA genes ofM. bovis BCG (19) andthe 16S rRNA genes from various other procaryotic speciesdescribed by Dams et al. (5) were selected and used for

EcoRI

synthesizing the oligonucleotides R (5'-GTGCCAGCAGCCGCGGTAA) and S (5'-GTGCGGGCCCCCGTCAATTC).These oligonucleotides correspond to residues 504 to 523and 910 to 929 on the opposite strands of the M. bovis BCGgene, respectively.

Polymerase chain reaction. The polymerase chain reaction(PCR) (17) was performed with thermoresistent DNA poly-merase from Thermus aquaticus (Taq polymerase; ThePerkin-Elmer Corp., Norwalk, Conn.) as recommended bythe manufacturer. One unit of Taq polymerase was added to100 ,ul of 50 mM NaCl-2 mM MgC12-10 mM Tris hydrochlo-ride-0.01% (wt/vol) gelatin-0.2 mM of each of the deoxynu-cleotides dGTP, dATP, dTTP, and dCTP (BoehringerGmbH, Mannheim, Federal Republic of Germany) (pH 9.6),

XhoI

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FIG. 1. Physical map of the 4.6-kb EcoRI insert of pRP5000 containing IS986 flanked by inverted repeats (IR). Also, the large 1,037-bpopen reading frame (ORF) is depicted.

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FIG. 2. Southern blot analysis of PvuII-digested chromosomal DNA of various mycobacterial species hybridized with the 32P-labeled4.6-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. gordonae71; lanes 19, M. intracellulare 56; lanes 20, M. kansasii 53. Numbers on the left indicate sizes of standard DNA fragments in kilobase pairs.

VOL. 28, 1990

2054 HERMANS ET AL.

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FIG. 3. Southern blot analysis of PvuIl-digested chromosomal DNA of various mycobacterial species with the 32P-labeled 386-bpBamHI-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; lane15, 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 theleft 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 2min, 72°C. The amplified DNA was analyzed by agarose gelelectrophoresis.Animal experiments. TRL guinea pigs (weight, about 250

g) were subcutaneously and intramuscularly inoculated inone leg with 0.5 ml of a M. tuberculosis suspension of 102bacilli per 1 ml of phosphate-buffered saline. After 8 weeks,the animals were sacrificed, M. tuberculosis was isolatedfrom the spleens, and the bacteria were further cultured invitro.

RESULTSOccurrence of IS986 among the various mycobacterial

species. In order to investigate the occurrence of IS986 inmycobacteria, plasmid pRP5000 was used for the isolation ofthe insertion element. pRP5000 is a derivative of a lambdarecombinant that was selected from a M. tuberculosis genelibrary as a result of its hybridization to the M. fortuitumplasmid pUS300 (24). The physical map of the mycobacterialinsert of pRP5000 is shown in Fig. 1. The 4.6-kb EcoRI insertwas isolated and hybridized with PvuII-digested genomicDNA from different mycobacterial species. Multiple hybrid-izing 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. chelo-nei, M. smegmatis, or M. perigrinum (data not shown).These results indicate that the occurrence of the IS986-likeelements is restricted to strains belonging to the M. tuber-culosis complex group.Polymorphism of restriction fragments containing IS986.

All 14 M. tuberculosis complex strains shown in Fig. 2Acontained multiple DNA fragments which hybridized withthe 4.6-kb insert of pRP5000, which is consistent with theprevious findings of Zainuddin and Dale (24). Furthermore,each strain displayed a different, unique banding pattern. Todemonstrate that this RFLP is a special property of thefragments containing the IS986 element, we rehybridized thesame blot with the 2.4-kb EcoRI fragment of pPH7301. Thisplasmid contains another repeated sequence that is presentin the M. tuberculosis complex species M. gordonae and M.kansasii (12a). In contrast to the hybridization patternsobtained with the 4.6-kb EcoRI fragment of pRP5000, virtu-ally identical banding patterns were observed among thedifferent M. tuberculosis complex strains (Fig. 2B). Se-quence data showed that IS986 is located between twoBspMII sites (McAdam et al., submitted) which are uniquesites in the inverted repeats of this insertion sequenceelement (Fig. 1). To confirm that the RFLP is due torestriction fragments that contain IS986, we used the 386-bpBamHI-XhoI fragment located within the insertion elementof pRP5000 as a probe for the hybridization of PvuII-digested chromosomal DNA of the same mycobacterial

J. CLIN. MICROBIOL.

TOOL FOR DIAGNOSIS AND EPIDEMIOLOGY OF TUBERCULOSIS

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FIG. 4. RFLP analysis ofM. bovis BCG strains. M. tuberculosis15 (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. bovisBCG 45 (lane 6), M. bovis BCG 43 (lane 7), M. bovis BCG 105 (lane8), and M. bovis BCG 106 (lane 9) were analyzed. Southern blottingwas performed as described in the legend to Fig. 3. Numbers on theleft indicate sizes of standard DNA fragments in kilobase pairs.

strains used in Fig. 2. As expected, this small fragment didnot hybridize with species other than those of the M.tuberculosis complex. The banding patterns obtained withthe M. tuberculosis complex strains were similar but notidentical to those obtained with the 4.6-kb EcoRI probe (Fig.3). This is consistent with the idea that the RFLP is due tothe presence of IS986 at different sites in the genome of thevarious 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 the386-bp IS986-specific DNA probe. This indicates that IS986is present as a single copy in this clinical isolate. Weexamined six additional M. bovis BCG strains, both vaccinestrains as well as clinical isolates. All M. bovis BCG strainstested were found to contain a unique 1.7-kb PvuII fragmentthat hybridized with the 386-bp fragment IS986 (Fig. 4). Weconclude that IS986 is present as a single copy in all M. bovisBCG strains and that it is inserted at the same location in thegenome.

Specific DNA amplification of IS986 by PCR. Based on thesequence of IS986 (McAdam et al., submitted), two oligo-nucleotides, INS-1 and INS-2, were synthesized. Theseoligonucleotides were used as primers for amplification byPCR of chromosomal DNA corresponding to residues 641 to885 of IS986. As expected, after amplification of either M.tuberculosis DNA or pRP5000, a fragment of approximately245 bp was visible on agarose gels (Fig. 5A). ChromosomalDNAs of M. africanum, M. bovis, M. bovis BCG, and M.microti were also found to contain the 245-bp amplifiablefragment. Furthermore, chromosomal DNAs from 22 M.

FIG. 5. Agarose gel electrophoresis of amplified DNAs of dif-ferent mycobacterial strains by using oligonucleotide amplimer pairsINS-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; lanes10, M. scrofulaceum 65; lanes 11, M. smegmatis 67; lanes 12, M.leprae 90. Numbers on the left indicate sizes of standard DNAfragments in base pairs.

tuberculosis strains, 6 M. bovis BCG strains, and 1 M. bovisstrain were tested by PCR; and all were shown to contain the245-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 invitro, the two oligonucleotide primers R and S, which arehomologous to the conserved regions of the procaryotic 16SrRNA genes (5, 19), were used to test the genomic DNAisolates. This resulted in the amplification of a 426-bpfragment from all mycobacterial species mentioned above(Fig. 5B).

Genetic stability of IS986 during animal passage. The highdegree of RFLP in IS986-containing sequences of M. tuber-culosis complex strains suggests that this insertion elementcould transpose with a high frequency. To investigatewhether genetic rearrangement of IS986 occurs after passagein animals susceptible to M. tuberculosis, we analyzed therestriction fragment patterns of four strains before and afterpassage during 2 months in guinea pigs. The results areshown in Fig. 6. No changes in the banding pattern were

VOL. 28, 1990 2055

2056 HERMANS ET AL.

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observed, indicating that IS986 is stable during a limitednumber of generations in vivo.Use of IS986 for epidemiological purposes. The observed

RFLP caused by IS986 suggests the possibility that IS986can be used for precise epidemiological investigations.Therefore, we tested an additional 15 clinical isolates of M.tuberculosis. These isolates were obtained from variousregional hospitals and laboratories in The Netherlands. Atotal of 6 of the 15 strains were not known to originate fromepidemiologically related cases of tuberculosis, and thesestrains showed different PvuII banding patterns when the386-bp IS986 probe was used (data not shown). This is inmarked contrast to the remaining nine isolates, which exhib-ited a mutually identical restriction fragment pattern (Fig. 7,lanes 1 to 9). These nine isolates originated from an outbreakof tuberculosis among individuals who were all treated bythe same physician, who specialized in the treatment ofpatients with arthritis. The identities of the banding patternsfrom these isolates from a known outbreak indicate thepotential value of IS986 as a probe for such studies. Furtherconfirmation of the usefulness of this probe was provided bya study of 26 M. tuberculosis isolates which were obtainedfrom a single regional health laboratory in January 1990; atthat 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 theycould be traced to the same outbreak. An additional threestrains (Fig. 8, lanes 15, 24, and 25) also showed character-

FIG. 7. RFLP analysis of nine strains from an M. tuberculosisoutbreak (lanes 1 to 9) and two nonrelated strains (lanes 10 and 11).M. tuberculosis 92 (lane 1), M. tuberculosis 93 (lane 2), M. tuber-culosis 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. tubercu-losis 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 inkilobase pairs.

istic banding patterns which were not related to the patternsof the strains involved in the outbreak. Case histories ofthese patients revealed that they were suspected of beingcontacts of a common source of infection. The application ofthe probe in this case was therefore successful in revealing apreviously unsuspected cluster of cases of tuberculosis.

DISCUSSION

As repetitive segments of DNA are found in virtually allprocaryotic and eucaryotic organisms (8), it is not surprisingthat these elements have recently also been identified inmycobacteria. Clark-Curtiss and colleagues (2, 3, 12) founda 2.2-kb sequence that was present in at least 19 copies perM. leprae genome but that was not present in other myco-bacterial species. Another species-specific repetitive DNAelement in M. paratuberculosis was identified by Green et al.(11). This repeated sequence has significant homology withISJlO, an insertion element of Streptomyces coelicolor.Various strains of M. leprae and M. paratuberculosis havebeen analyzed for RFLP by using the repetitive DNA as aprobe, and the nonvariability of the banding patterns indi-cated that these elements were inserted at a specific positionin the Mycobacterium chromosome. In M. tuberculosis,repetitive DNA elements have been found that are insertedat a specific location in the genome (12a), whereas otherswere 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 (lane22), 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 ofstandard DNA fragments in kilobase pairs.

the putative insertion sequence IS986. This element is 1,386bp in size and is flanked by inverted repeats that arehomologous to those of IS3 and IS3411. Furthermore, IS986contains an open reading frame, which encodes a putativetransposase, which is significantly homologous in its aminoacid sequence to the transposases of the IS3 family ofenterobacterial insertion elements (McAdam et al., submit-ted). A similar but not identical element was recently se-quenced by Thierry et al. (20).

In this study, we showed that IS986-like elements arepresent in 1 to 20 copies per genome and that these elementsare specific for the species of the M. tuberculosis complex.We extend the preliminary observations of Zainuddin andDale (24) that these elements differ in their chromosomallocation from strain to strain. The patterns of the restrictionfragments containing IS986 in all 32 independent isolates ofM. tuberculosis strains were different. Such an extremedegree of polymorphism reinforces the idea that IS986 is afunctional insertion element that can insert to new sites onthe same replicon and that can initiate other types of geneticrearrangements such as cointegration of replicons, inver-sion, and deletion of sequences adjacent to the insertionsequence element (8). The latter type of genetic event hasrecently been suggested by Hermans et al. (12a) to be aconsequence of the activity of another repetitive DNAelement in M. tuberculosis. This study shows that the highdegree of polymorphism of IS986-containing fragments isvery useful as a tool in determining the epidemiology oftuberculosis. All strains from an epidemiologically well-defined outbreak of tuberculosis were of the same charac-teristic RFLP type. Furthermore, in a collection of 26 strainsobtained from a peripheral laboratory in the southern part ofThe Netherlands, we could identify two strains with thisRFLP pattern. Later, it appeared that these strains origi-

nated from patients involved in the same outbreak men-tioned above. Also, three strains with another characteristicRFLP pattern were recognized, and these strains weresuspected of being contacts with individuals involved in asingle tuberculosis outbreak. We are in the process ofdetermining the RFLP types of all M. tuberculosis strainsthat are sent to our reference laboratory. By computeranalysis of a library of previously established RFLP pat-terns, one might be able to trace sources of M. tuberculosisfrom the distant past.An interesting observation was the nonpolymorphic pat-

tern of IS986-containing restriction fragments of M. bovisBCG. This derivative was obtained in 1921 from a patho-genic Mycobacterium strain that lost its virulence graduallyduring passages in liquid medium (1). Since then, manysubstrains of M. bovis BCG have been used to preparevaccines around the world, and these substrains are hetero-geneous in many of their characteristics. As all strainsinvestigated in this study were of the same RFLP type andcontained a single copy of IS986 in their genomes, all M.bovis BCG isolates are likely to contain a single IS986 copyat one particular location in the chromosome. This suggeststhat IS986 lost its transposition capability before or duringthe 230 passages of the parental strain of M. bovis BCG andthat all substrains are identical with respect to the site ofinsertion of IS986 in the chromosome. This unique locationin all M. bovis BCG strains will be useful in distinguishing M.bovis BCG from other mycobacteria. We will investigatewhether there is a correlation between the loss of virulenceof M. bovis BCG and the location of IS986 in the chromo-some. The high polymorphism of fragments containing IS986among clinical isolates of M. tuberculosis complex strainssuggests either that IS986 changes frequently in its positionin the chromosome or that IS986 is relatively stable but that

VOL. 28, 1990 2057

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2058 HERMANS ET AL.

the clinical isolates examined in this study have a longhistory of separate lineage. Our observations that no poly-morphism was observed among strains from single out-breaks and that serial passage in guinea pigs does not lead tosubstrains with IS986 at a different chromosomal locationpoint toward the latter possibility and suggest that IS986 is arelatively stable element which transposes with a low fre-quency.Because IS986 is found only in M. tuberculosis complex

strains, this element is a useful target for in vitro amplifica-tion by PCR for the rapid and specific detection of suchbacteria in clinical specimens, without the need for culturingthese slowly growing microorganisms.

ACKNOWLEDGMENTS

We thank T. van der Werf, P. L. van Putten, and F. Portaels forproviding us with mycobacterial strains and J. G. Baas for helpfuldiscussions and technical assistance.

This study received financial support from the World HealthOrganization Programme for Vaccine Development.

LITERATURE CITED1. Calmette, A., L. Negre, and A. Boquet. 1922. Essais de vacci-

nation 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-specificrepetitive sequence in Mycobacterium leprae DNA. J. Infect.Dis. 159:7-15.

3. Clark-Curtiss, J., and G. P. Walsh. 1989. Conservation ofgenomic sequences among isolates of Mycobacterium tubercu-losis. J. Bacteriol. 171:4844-4851.

4. Crawford, J., and J. H. Bates. 1984. Phage typing of mycobac-teria, p. 123-132. In G. P. Kubica and L. G. Wayne (ed.), Themycobacteria: a sourcebook. Part A. Marcel Dekker, Inc., NewYork.

5. Dams, E., L. Hendriks, Y. Van de Peer, J. Neefs, G. Smits, I.Vandenbempt, and R. De Wachter. 1988. Compilation of smallribosomal 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 tuber-culosis. 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 se-quences, 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. Forensicapplication ofDNA 'fingerprint.' Nature (London) 318:577-579.

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