Mycobacterium tuberculosis

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1993, p /93/ $02.00/0 Copyright C 1993, Americn Society for Microbiology Vol. 31, No. 8 Comprison of Vrious Repetitive DNA Elements s Genetic Mrkers for Strin Differentition nd Epidemiology of Mycobcterium tuberculosis DICK van SOOLINGEN,' PETRA E. W. DE HAAS,' PETER W. M. HERMANS,2 PETER M. A. GROENEN,' AND JAN D. A. VAN EMBDENl* Unit Moleculr Microbiology nd Lbortory of Bcteriology, Ntionl Institute of Public Helth nd Environmentl Protection, P.O. Box 1, 3720 BA Bilthoven, The Netherlnds, 1 nd Armuer Hnsen Reserch Institute, Addis Abb, Ethiopi2 Received 29 Mrch 1993/Accepted 4 My 1993 Five different genetic elements hve been found to be ssocited with genetic rerrngements in Mycobcterium tuberculosis complex strins. Of these elements, the insertion sequence IS6110 is presently the most frequently used genetic mrker for strin differentition ofm. tuberculosis. In the present study we compred five genetic elements for their potentils to differentite given cluster ofm. tuberculosis strins. Becuse of the presence of only single copy of IS6110 or two IS6110 copies t the sme chromosoml locus, lrge number of strins could not be differentited by IS6110 fingerprinting. Most strins, including the low-copy-number IS6110 strins, could be differentited by fingerprinting with the 36-bp direct repet or the polymorphic GC-rich repetitive DNA element. Less discrimintive power ws obtined with the mjor polymorphic tndem repet nd the insertion element IS1081. One strin which did not contin IS6110 DNA ws encountered. Until now, this element hs invrintly been found to be present in ll M. tuberculosis complex strins. On the bsis of clssicl txonomic criteri nd sequencing of the 16S rrna gene, this strin ws shown to be genuline M. tuberculosis strin. Therefore, the use of this element s trget for polymerse chin rection-fcilitted detection of M. tuberculosis should be reconsidered. Since the discovery of polymorphic DNA in Mycobcterium tuberculosis, strin differentition hs become n importnt tool in the epidemiology of tuberculosis. Among the vrious genetic elements tht hve been found to contribute to DNA polymorphism in M. tuberculosis, the insertion element IS6110 hs been studied most intensively (8, 15, 25, 32). This 1,355-bp insertion sequence (IS) element is relted to the IS3 fmily of ISs nd hs been found exclusively in ll isoltes of the M. tuberculosis complex group of mycobcteri (12, 17, 25). Becuse of the highly vrible number of copies nd the vrible sites of integrtion in the chromosome, epidemiologiclly unrelted strins disply n extremely high degree of polymorphism of IS6110-contining restriction frgments (3, 12, 14, 16). This polymorphism is presumbly due to the bility of IS6110 to trnspose within the genome without much trget sequence specificity (11, 12, 16, 17). Despite the high degree of IS6110-ssocited restriction frgment length polymorphism (RFLP) mong M. tuberculosis strins, no IS6110 trnsposition hs been observed during prolonged in vitro culturing or in vivo growth (12, 18), suggesting tht the frequency of trnsposition is reltively low. This is in greement with observtions which indicte tht IS6110 is n excellent genetic mrker for trcing individul M. tuberculosis strins during microepidemics, nosocomil infections, nd the clonl dissemintion of multipledrug-resistnt strins (1, 2, 5-7, 9, 10, 12, 14, 17, 18, 22, 25, 29) Ȧlthough most M. tuberculosis complex strins crry multiple IS6110 copies, isoltes which contin only one or few copies hve occsionlly been encountered. Virtully ll Mycobcterium bovis BCG strins contin single IS copy * Corresponding uthor in region of the chromosome in which multiple short directly repeted sequences re present. Becuse the mjority of M. tuberculosis complex strins lso crry one or two copies of the IS element t this chromosoml locus, this site seems to be preferred site for integrtion of IS6110 (11). During preliminry nlysis of strins originting from Indi, we encountered lrge number of strins with only single IS copy. These strins could not be differentited by IS6110 DNA fingerprinting, becuse IS6110 DNA ws found to be ssocited with unique restriction frgment in these strins. As expected, IS6110 ws found to be inserted in the region contining the direct repets (DRs). Becuse to our knowledge these isoltes originted from epidemiologiclly unrelted cses of tuberculosis, we wondered whether such strins might be differentited by other polymorphic DNA mrkers. In ddition to IS6110, four other trgets for RFLP nlysis hve been described: (i) IS1081, n insertion element relted to the Stphylococcus ureus trnsposble element IS254 (4, 30); (ii) polymorphic GC-rich repetitive sequence (PGRS) present on the recombinnt plsmid TBN12 (20); (iii) the mjor polymorphic tndem repet (MPTR) sequence (13); nd (iv) the 36-bp DR sequence of which multiple copies re present t the hot spot of IS6110 integrtion (11). The cluster of DR sequences is present t single chromosoml locus, wheres the other four elements re locted t mny different positions in the chromosome. Although ech of these five repetitive DNA sequences hs been shown to be implicted in RFLP in M. tuberculosis, no ttempt hs been mde to compre the RFLPs of ech of these genetic mrkers in M. tuberculosis. The study described here ws undertken to determine which of these five polymorphic genetic mrkers ws most suitble for strin differentition of M. tuberculosis, especilly strins contining few IS6110 copies. We

2 1988 van SOOLINGEN ET AL. J. CLIN. MICROBIOL. Kpn I Pvull DR IS Kpn I-Pvu 11 probe (left rm) 245 bp probe (right rm) INSi nd JNS2 primers FIG. 1. Physicl mp of the M. bovis BCG chromosoml region contining the IS6110-like element IS987. The DRs re depicted s smll, numbered blocks. The DRs re spced by unique intervening sequences of 35 to 41 bp in length (11). The probes nd primers used in the present study re indicted. showed tht epidemiologiclly nonrelted strins which cnnot be differentited with IS6110 becuse of the presence of only one or few copies cn be distinguished by other genetic mrkers, in prticulr, by DR nd PGRS DNAs. Furthermore, we describe strin which is exceptionl in tht it lcks IS6110 DNA nd show by 16S rrna gene sequencing nd other methods tht this strin is genuine M. tuberculosis strin. MATERIALS AND METHODS Bcteril strins nd bcteriologicl testing. Sixty-three strins originting from Indi were obtined in 1984 from D. W. Smith (University of Wisconsin, Mdison). M. tuberculosis strins isolted in the People's Republic of Chin were supplied by Wng Guozhi nd Wng Zheng (Ntionl Institute for the Control of Phrmceuticl nd Biologicl Products, Beijing, People's Republic of Chin). Strins crrying single IS6110 copy isolted in The Netherlnds were sent to us by Regionl Helth Lbortories. Other strins with single copies of IS6110 were kind gifts of R. Hussin (Ag Khn University, Krchi, Pkistn), T. Aisu (Ugnd Tuberculosis Investigtions, Wndegey Kmpl, Ugnd), P. M. Smll (Howrd Hughes Medicl Institute, Stnford, Clif.), nd J. Perer (University of Colombo, Colombo, Sri Lnk). Bcteriology bsed on biochemicl nd growth chrcteristics nd sensitivity testing of guine pigs for pthogenic mycobcteri were performed s described previously (23, 31). Ftty cid nlysis of mycobcteril cells ws done s described by Tisdll et l. (26). The rectivity of mycobcteri with the M. tuberculosis complex-specific monoclonl ntibody F42-2 ws determined s described by Schoningh et l. (21). DNA techniques. Genomic DNA extrction nd Southern blotting were performed s described previously (27-29). DNA frgments for Southern blot hybridiztion were lbeled by using the enhnced chemiluminescence gene detection system (Amershm Interntionl plc, Amershm, United Kingdom). Oligonucleotides were lbeled by using the ECL 3' oligo lbeling system (Amershm Interntionl plc). Two different IS6110 probes were used in the present study: 245-bp product mplified by polymerse chin rection (PCR) (IS6110R) (12) nd KpnI-PvuII frgment of IS6110 (IS6110L) (12). The ltter DNA ws purified by grose gel electrophoresis of KpnI-PvuII-cleved pph1001 plsmid DNA (11). Plsmid ptbn12 (20) ws kind gift of B. Ross (Firfield Infectious Disese Hospitl, Firfield, Austrli). This plsmid contins cluster of short PGRSs, nd the plsmid ws used in its entirety for hybridiztion experiments. IS1081 DNA ws obtined by PCR mplifiction s described previously (30). The probe DR-r, which ws used to detect the direct repets in the hot spot integrtion region of IS6110, hs been described previously (11). As probe for the MPTR, we used the synthetic oligonucleotide TR2 (13). Gen-Probe tests were performed ccording to the recommendtions of the mnufcturer (Gen-Probe Incorported, Sn Diego, Clif.). The 5' 550-bp region of the 16S rrna gene ws mplified by PCR by using the brod-host-rnge primers 16S1F, AGAGT1TTGATC(A/C)TGG(T/C)TCAG, nd 16S1R, C-L'TACGCCCA(A/G)T(G/A)A(ATI)TCCG. The PCR product ws sequenced on n utomtic sequencer (model 370A; Applied BioSystems). RESULTS Determintion of DNA polymorphism mong M. tuberculosis strins by using five different genetic mrkers. In order to compre the bility of the five DNA sequences to detect DNA polymorphism mong M. tuberculosis strins, these DNA sequences were used s probes in RFLP typing of 63 strins isolted in southern Indi. The IS6110 fingerprints obtined with the "right rm" probe IS6110R (Fig. 1) of this set of strins re shown in Fig. 2. Although the mjority of the M. tuberculosis strins differed in their IS6110 bnding ptterns, 21 of 63 strins hrbored single IS6110 copy, nd in 20 of these strins, the IS element ws found to be present on 1.5-kb PvuII restriction frgment. In contrst, none of the DNA fingerprints of the strins crrying multiple copies of IS6110 were identicl. Unexpectedly, the DNA of one M. tuberculosis strin, strin IN4, did not hybridize with the IS6110 probe (Fig. 2B). This indictes tht t lest prt of the IS6110 DNA is bsent from this strin. We selected the 22 M. tuberculosis strins used for the DNA fingerprinting shown in Fig. 2B for comprtive nlysis with other DNA probes. This subset contined mny strins with single, identiclly sized IS6110-contining PvuII frgment, in ddition to strins with highly relted RFLPs. The filter contining chromosoml PvuII frgments of this subset ws rehybridized with the lbeled KpnI-PvuII frgment of IS6110 (the "left rm," IS6110L; Fig. 1), IS1081, PGRS, DR, nd MPTR DNAs. The DNA fingerprints of seven M. tuberculosis strins showing single 1.5-kb frgment hybridizing with the IS6110R probe lso showed single, unique PvuII frgment when IS6110L ws used s probe (Fig. 3A). This strongly suggests tht the IS element in these strins is inserted t unique position in the chromosome. The fingerprints obtined with IS1081 were much less polymorphic compred with those obtined with IS6110

3 ~~~ ~ - 4,~~ 4S..~w ~ ~ ~~~0~ ~ VOL. 31, 1993 M. TUBERCULOSIS DIFFERENTIATION AND EPIDEMIOLOGY 1989 Lne Indi strin A Lne Indi B Lne Indi C strin strin ;,q, 1. 4s so e i M m " 4 OM 1,1 - - mftmmm ; U -S ~ n - Si e~ ~~-0 A.S -em e m m% N m - m - 1M4" m 0-4m FIG. 2. DNA fingerprints of 63 M. tuberculosis strins originting from Indi. Chromosoml DNA ws digested with the restriction enzyme PvuII. The 245-bp PCR frgment (IS6110R) of IS6110 ws used s probe to visulize PvuII restriction frgments contining the right rm of IS6110. Numbers on the left indicte the sizes of stndrd DNA frgments (in kilobse pirs). Lne 22 of ech pnel represents DNA of M. tuberculosis reference strin Mt

4 Lne Indi strin A ~~~~~~~~~~~~~~. m - g w Wl omm B C w srn- S -SW l1. m4e -- f l m e mi '4 P. --. m "t m.q.. 41~ omq ~W go - -p flw wo AM.. w. D ' o-w A FIG. 3. RFLP of M. tuberculosis DNA by using the left rm of IS6110 (A), IS1081 (B), DR (C), nd PGRS DNA (D) s probes. T'he strins nlyzed re identicl to those in Fig. 2B. (A nd B) Fingerprints from PvuII-digested DNAs; (C nd D) fingerprints fromalul-digested DNAs. Numbers on the left indicte sizes of stndrd DNA frgments (in kilobse pirs). In ll pnels, lne 22 ws loded with the DNA of M. tuberculosis reference strin Mt

5 VOL. 31, 1993 M. TUBERCULOSIS DIFFERENTIATION AND EPIDEMIOLOGY 1991 (Fig. 3B). All 22 strins crried either six or seven IS1081 copies, nd only six different ptterns were observed. This limited polymorphism is consistent with our previous observtions on the IS1081 RFLP mong other M. tuberculosis strins. A poor differentition between fingerprints ws obtined with the remining three DNA probes becuse the MPTR nd the PGRS probes hybridized to mny PvuII restriction frgments, resulting in lmost identicl overcrowded bnding ptterns nd becuse DR DNA hybridized to only few frgments tht differed only slightly in size (dt not shown). Therefore, chromosoml DNAs of this subset were cleved with nother restriction enzyme, AluI, which cleves more frequently thn PvuII. Agin, little strin differentition ws obtined with the MPTR probe (dt not shown); however, the ptterns obtined with PGRS nd DR probes were more polymorphic compred with the ptterns obtined with the PvuII frgments. Figure 3D shows the fingerprints obtined with PGRS DNA s probe. A vst number of AluI PGRS-hybridizing frgments re visible in Fig. 3D, nd the RFLP ws most conspicuously visible mong frgments lrger thn 3 kb. The bnding ptterns of ll 22 strins were different, including those from strins with single IS6110 element. The DR fingerprints re shown in Fig. 3C. Twenty different AluI ptterns could be distinguished mong the 22 M. tuberculosis strins. All except two of the seven strins with single IS6110 copy were differentited by the DR probe (Fig. 2B nd 3C, lnes 10 nd 13). The other strin pir with n identicl DR fingerprint ws from group of strins tht displyed closely relted IS6110 fingerprints (Fig. 2B, 3A, nd 3C, lnes 4 nd 11). It should be noted tht the 1.5-kb PvuII IS6110-contining frgment in the strins with single copy of IS6110 cohybridized with DR DNA, indicting tht the IS6110 element is inserted in the DR region of the chromosome. We conclude tht the DR nd the PGRS probes llow differentition of strins which re indistinguishble by the IS6110 probe becuse of the IS6110 insertion into unique position in the chromosome. DNA polymorphism in the DR cluster of epidemiologiclly nonrelted strins with identicl IS6110 fingerprints. The results described bove indicte tht M. tuberculosis strins with n identicl IS6110 DNA fingerprint re not necessrily identicl, becuse the strins with single IS6110 copy locted on 1.5-kb PvuII frgment generlly show different fingerprint when probed with PGRS or DR DNA. Previously, we hve observed the occurrence of M. tuberculosis strins contining single IS6110 element (13, 28, 29, 30). In the mjority of these strins, the IS element ws found to be present t 1.5-kb PvuII frgment, wheres most of these strins originted from epidemiologiclly unrelted cses of tuberculosis. To investigte more systemticlly the DNA polymorphism of such strins by using other genetic mrkers, we subjected ll 21 single-is6110-copy strins from the set of 63 strins from Indi described bove nd 16 single-is6110- copy strins from six other countries to RFLP nlysis using the DR probe. In 29 of these 37 strins, the IS6110R probe hybridized with PvuII frgment of 1.5 kb, nd in the remining strins the IS6110R probe hybridized with either 1.3- or 5.0-kb frgment. The results of the DR DNA hybridiztion experiment re shown in Fig. 4. A high degree of AluI RFLP ws lso observed mong the mjority of these strins. Thirty-four different DR-contining frgment ptterns were distinguished mong the 37 strins investigted. This indictes tht the mjority of these strins re different, which is consistent with the fct tht these strins were supposed to be epidemiologiclly unrelted. In previous report (29), we hve described n outbrek of tuberculosis mong homeless people in Amsterdm, nd the vst mjority of the strins from tht outbrek showed n IS6110 fingerprint consisting of two PvuII bnds with sizes of 2.9 nd 2.1 kb. Strins with this chrcteristic bnding pttern hve been encountered repetedly since then, lthough no pprent epidemiologicl reltionship with the outbrek ws suspected. To investigte whether the ltter strins were identicl to the ones involved in the outbrek in Amsterdm, we nlyzed by DR typing 11 outbrek strins nd seven strins isolted elsewhere in The Netherlnds 1 to 2 yers fter the outbrek in Amsterdm. All 11 outbrek strins nd 3 other strins were identicl by DR fingerprinting, wheres the remining 4 strins clerly differed from the outbrek strins. Of the ltter four strins, two showed identicl DR fingerprints, wheres the other two hd different ptterns (dt not shown). Among the sme 18 strins, ech of which crried only two copies of IS6110, DNA fingerprints were lso produced by using the PGRS probe. Strins shring identicl DR DNA fingerprints lso ppered to hve the sme PGRS ptterns, wheres the two strins tht were epidemiologiclly unrelted but tht shred the sme DR DNA fingerprint lso hd identicl PGRS ptterns (dt not shown). We conclude tht the DR nd the PGRS probes re eqully suitble for differentiting M. tuberculosis strins, which re difficult to distinguish with the IS611OR DNA probe becuse of the presence of single IS6110 element in the DR region. Comprison of IS6110 nd PGRS DNA typing of M. tuberculosis strins crrying multiple IS6110 copies. Previous studies hve shown tht outbrek strins re identicl by IS6110 typing (12, 29). To determine whether this is lso true for PGRS typing, we nlyzed 12 isoltes from previously described outbrek (12) nd 2 strins with the sme IS6110 type isolted 2 yers fter the min microepidemic. All 14 strins exhibited identicl Alul PGRS bnding ptterns (dt not shown). Furthermore, seven strins from recent microepidemic which hd identicl IS6110 fingerprints were lso found to hve the sme PGRS fingerprints (dt not shown). These dt indicte tht outbrek strins cn be well recognized by the PGRS DNA probe nd tht PGRS DNA restriction frgment ptterns hve enough stbility to be used s effective tools in the epidemiology of tuberculosis. Previous studies suggested tht IS6110 fingerprints from epidemiologiclly unrelted isoltes in countries with high rte of tuberculosis trnsmission re more similr thn re such strins from countries with low trnsmission rte (29). To compre the discrimintive power of IS6110 with PGRS DNA for strins from res of high endemicity, we nlyzed set of 18 M. tuberculosis strins originting in the People's Republic of Chin. As shown in Fig. SA, the IS6110 bnding ptterns showed much reltedness, but ech pttern ws distinct. The AluI PGRS bnding ptterns lso showed much reltedness, nd only 13 different ptterns were observed mong the 18 strins. Identicl fingerprints were found mong four strins (Fig. 5B, lnes 4, 11, 12, nd 15) nd two pirs of strins (Fig. SB, lnes 5 nd 16 nd lnes 7 nd 9). Finlly, to investigte the chnges in the IS6110 nd PGRS DNA bnding ptterns of isogenic M. tuberculosis strins which hve been cultured seprtely for mny yers, the lbortory strins H37Rv nd its virulent derivtive H37R

6 1992 van SOOLINGEN ET AL. J. CLIN. MICROBIOL. Lne Indi strin A m , n.. S. Om Wm mm Lne Country. B N N N N N P P Ug Ug E N N N N Us S IEmm, - Om-- -1w m -- 4U* - O m.s FIG. 4. DR RFLP of M. tuberculosis strins crrying single IS6110 element. All DNAs were digested with the restriction enzyme AluI. The strins shown in pnel A re from Indi, nd the strins in pnel B originted from The Netherlnds (N), Pkistn (P), Ugnd (Ug), Ethiopi (E), the United Sttes (Us), nd Sri Lnk (S). Numbers on the left indicte the sizes of stndrd DNA frgments (in kilobse pirs). Lne 22 of pnel A contins DNA of M. tuberculosis reference strin Mt In these strins, IS6110R DNA is present on PvuII frgment of either 1.5 kb (pnel A, lnes 1 to 13 nd lnes 15 to 21; pnel B, lnes 1 to 8 nd 16), 1.3 kb (pnel B, lnes 11, 12, nd 15), or 5.0 kb (pnel A, lne 14; pnel B, lnes 9, 10, 13, nd 14) (dt not shown). were compred. The IS6110 fingerprints of these strins differed in 5 of 15 bnds, wheres the only difference between the PGRS ptterns ws one dditionl bnd in the lne of H37Rv (dt not shown). Chrcteriztion of n M. tuberculosis strin without IS6110 DNA. As described bove, DNA extrcted from one of the M. tuberculosis strins originting from Indi did not hybridize with IS6110 DNA. Also, by PCR with the INS1 nd INS2 primers (Fig. 1), we found no mplifible IS6110 DNA in strin IN4. Strin IN4 is exceptionl mong M. tuberculosis strins becuse this ws the first strin without detectble IS6110 DNA mong t lest 700 M. tuberculosis complex strins tested so fr. Hybridiztion experiments with other genetic mrkers reveled the presence of DR, PGRS, nd IS1081 DNAs in the genome of this exceptionl strin (Fig. 3B, C, nd D). Becuse this strin might lso differ from M. tuberculosis in other chrcteristics, strin IN4 ws subjected to thorough bcteriologicl identifiction procedure. The Gen-Probe test for M. tuberculosis complex strins ws positive with strin IN4. Becuse this test is bsed on the hybridiztion of only 20-bp stretch of the 16S rrna.o U-A sequence, we sequenced the 5' 550 bp of the 16S rrna gene in strin IN4. The sequence obtined ws identicl to the ones previously published for M. tuberculosis nd M. bovis BCG (19, 24) (EMBL dt bse ccession numbers X52917 nd M20940, respectively). Strin IN4 ws lso subjected to clssicl bcteriologicl determintion, gs chromtogrphy of cell wll-ssocited lipids, nd drug susceptibility testing with n extended spectrum of ntibiotics. Agin, the results were chrcteristic for M. tuberculosis. Furthermore, strin IN4 ws rective with the monoclonl ntibody F24-2, which hs been shown to be specific for bcteri of the M. tuberculosis complex (21). Finlly, guine pigs showed the chrcteristic tuberculous lesions 2 months fter subcutneous inocultion with strin IN4. DISCUSSION Although five different genetic repetitive DNA elements hve been shown to be implicted in chromosoml DNA polymorphism of M. tuberculosis, only IS6110 nd PGRS DNA hve previously been tested with lrge number of

7 VOL. 31, 1993 M. TUBERCULOSIS DIFFERENTIATION AND EPIDEMIOLOGY 1993 A A 1: B : S ~ - ^~ LW.. -~ ~~~~~ - *, f' P.---.-,--- G iv S3*ffflg-ffl;3 2.0 p- FIG. 5. IS6110 (A) nd PGRS DNA (B) contining restriction frgment ptterns of M. tuberculosis strins originting from the People's Republic of Chin. An identicl set of strins ws nlyzed in both pnels. The DNAs in pnel A were digested with PvuII, nd the DNAs in pnel B were digested with AluI. Numbers on the left indicte the sizes of stndrd DNA frgments (in kilobse pirs). isoltes. The results of the present study confirm the conclusions of previous studies tht both IS6110 nd PGRS DNA re excellent mrkers for use in differentiting M. tuberculosis strins. Furthermore, the DR region ws lso found to be very polymorphic, llowing the differentition of virtully ll 63 Indin M. tuberculosis strins investigted in the present study. As observed previously (30), the IS1081 element ws found to be less useful for strin differentition becuse most of the five to seven copies of IS1081 re present t identicl positions in the M. tuberculosis chromosome. Similrly, the MPTR sequence llowed only limited strin differentition. Becuse IS6110 is the most frequently used genetic element for differentiting M. tuberculosis strins, it is of criticl importnce to know whether strins with identicl IS6110 bnding ptterns belong to single group of outbrek strins tht were recently disseminted in clonl wy. The present study showed tht when only one or few IS6110 copies re present in M. tuberculosis strins, such isoltes my pper to be polymorphic when other genetic mrkers re used. Hermns et l. (11) hve shown tht bout 85% of the M. tuberculosis isoltes tht they investigted contined one or two copies of IS6110 in the DR-contining region of the chromosome, which seems to be hot spot for integrtion of IS6110. We previously found tht in mny strins, IS6110 DNA is present on 1.5-kb PvuII frgment (29) nd tht this frgment usully contins DR DNA (11). Therefore, the finding tht 32 of the 63 Indin strins investigted in the present study crried IS6110 DNA on the 1.5-kb PvuII frgment ws not unexpected. Twenty of these strins contined single IS6110 element, nd consequently, these isoltes could not be differentited with either the left rm or the right rm of IS6110 s the DNA probe. In ll of these strins, IS6110 ws found to be integrted in the DR region of the chromosome. However, most of these strins were esily differentited by the PGRS nd DR DNA probes. Although no thorough studies on the stbility of PGRS nd DR DNAs in M. tuberculosis hve been done, these results strongly suggest tht the 20 strins do not belong to recently disseminted clone, which is consistent with the unlikeliness tht ll of these strins re epidemiologiclly relted. Moreover, we found tht 16 strins originting from six different countries differed in their PGRS nd DR restriction frgment bnding ptterns, lthough mjority of these strins contined single copy of IS6110 t 1.5-kb PvuII restriction frgment. It is interesting tht the DR-contining AluI frgment ptterns mong the Indin strins ppered to be less polymorphic compred with those mong the strins from other countries. This would suggest tht the Indin strins belong to n evolutionrily relted group of strins which is distinct from strins isolted on other continents. It should be noted tht M. tuberculosis strins with single IS6110 copy hve rrely been observed (1-3, 5-7, 10, 11, 12, 14, 16-18, 22, 25, 29, 30). However, in none of those studies were fingerprints of Asin strins investigted. Results of the present study suggest tht significnt proportion of the M. tuberculosis strins from the Fr Est contin single IS6110 element t the unique hot spot for integrtion of this mobile element. Recent studies hve lso shown the occurrence of such strins in Vietnm (21). We lso investigted few epidemiologiclly unrelted M. tuberculosis strins crrying two IS6110 elements t identiclly sized PvuII frgments. Different types could be distinguished by DR nd PGRS fingerprinting, indicting tht these were different strins nd tht perhps second preferred site of integrtion for IS6110 exists in the mycobcteril chromosome. Although we encountered mny strins with single IS6110 element, the generl experience is tht M. tuberculosis isoltes contin multiple IS6110 elements nd tht strins with the sme IS6110 fingerprints re epidemiologiclly relted. The results described bove indicte tht certin repetitive DNA elements of M. tuberculosis re more unstble thn other ones. This opens the interesting possibility tht these elements could be used s differentil moleculr clocks to determine the evolutionry distnces between M. tuberculosis strins. It is cler tht the pce of the IS1081 clock is slower thn tht of IS6110, DRs, nd PGRS DNA. However, very little is known bout the quntitive differences between the pce of genetic rerrngements medited by the remining repetitive DNA elements identified thus fr in M. tuberculosis. A complicting fctor in using IS elements for such purposes is undoubtedly the strong locus-dependent frequency of trnsposition, s suggested by the results of the present study. The observtion tht the power of the PGRS probe to discriminte strins with high multiplicity of IS6110 elements is less thn tht of IS6110 my indicte tht IS6110- ssocited rerrngements occur more frequently thn PGRS-ssocited ones do. However, the outbrek strins generlly were found to be identicl either by IS6110 typing or by using PGRS DNA. Repetitive DNA elements like the DR, MPTR, nd PGRS probes might be more useful thn IS elements for mesuring

8 1994 van SOOLINGEN ET AL. quntittive evolutionry reltionships, becuse the DNA rerrngements cused by these elements re likely to be more rndom in nture. This is becuse mny of the muttions cused by trnsposition of IS elements re expected to be fr from neutrl, in contrst to wht one might expect from vritions in short repetitive DNA sequences. Recent sequence dt of the DR region of vrious M. tuberculosis strins hve shown tht prt of the DNA polymorphism in this region cn be explined by homologous recombintion between the 36-bp DR sequences, which re spced by unique sequences 35 to 41 bp in length (10). Unexpectedly, we encountered for the first time strin which lcked demonstrble IS6110 DNA sequences. This strin originted from Indi, nd by ll criteri, it ws found to be genuine M. tuberculosis strin. If such strins would occur specificlly in certin geogrphic res, one should be reluctnt to use IS6110 DNA s trget for mplifiction by PCR for detection of M. tuberculosis in clinicl specimens. ACKNOWLEDGMENTS The technicl ssistnce of J. E. M. Pijnenburg nd J. G. Bs is grtefully cknowledged. We thnk E. H. Rozendl for photogrphic reproduction, L. M. Schouls nd A. M. C. Bergmns for 16S DNA gene sequencing, M. Dessens-Kroon nd E. C. Splburg for drug susceptibility testing, A. vn Silfhout for gs-liquid chromtogrphy, nd A. H. J. Kolk nd S. Kuijper for identifiction enzymelinked immunosorbent ssy. G. C. vn Krnen is cknowledged for secretril ssistnce. This study ws finncilly supported by the World Helth Orgniztion Progrmme for Vccine Development nd the Europen Community Progrm for Science Technology nd Development. REFERENCES 1. Beck-Sgue, C., S. W. Dooley, M. D. Hutton, J. Otten, A. Breeden, J. T. Crwford, A. E. Pitchenik, C. Woodley, G. Cuthen, nd W. R. Jrvis Hospitl outbrek of multidrug-resistnt Mycobcterium tuberculosis infections. JAMA. 268: Brin, R. E., J. I. Tokrs, M. H. Grieco, J. T. Crwford, J. Willims, E. M. Sordillo, K. R. Ong, J. 0. Kilburn, S. W. Dooley, K. G. Cstro, W. R. Jrvis, nd S. D. Holmberg An outbrek of multidrug-resistnt tuberculosis mong hospitlized ptients with the cquired immunodeficiency syndrome. N. Engl. J. Med. 23: Cve, M. D., K. D. Eisench, P. F. McDermott, J. H. Btes, nd J. T. Crwford IS6110: conservtion of sequence in the Mycobcterium tuberculosis complex nd its utiliztion in DNA fingerprinting. Mol. Cell. Probes 5: Collins, D. M., nd D. M. Stephens Identifiction of insertion sequence, IS1081, in Mycobcterium bovis. FEMS Lett. 83: Dley, C. L., P. M. Smll, G. F. Schecter, G. K. Schoolnik, R. A. McAdm, W. R. Jcobs, Jr., nd P. C. Hopewell An outbrek of tuberculosis with ccelerted progression mong persons infected with the humn immunodeficiency virus: n nlysis using restriction frgment length polymorphisms. N. Engl. J. Med. 326: Dooley, S. W., M. E. Villrino, M. Lwrence, et l Nosocomil trnsmission of tuberculosis in hospitl unit for HIV-infected ptients. JAMA 267: Edlin, B. R., J. I. Tokrs, M. H. Grieco, J. T. Crwford, J. Willims, E. M. Sordillo, K. R. Ong, J. 0. Kilburn, S. W. Dooley, K. G. Cstro, W. R. Jrvis, nd S. D. Holmberg An outbrek of multidrug-resistent tuberculosis mong hospitlized ptients with the cquired immunodeficiency syndrome. N. Engl. J. Med. 326: Eisench, K. D., J. T. Crwford, nd J. H. Btes Repetitive DNA sequences s probes for Mycobcterium tuberculosis. J. Clin. Microbiol. 26: Fischl, M. A., R. B. Uttmchndni, G. L. Dikos, et l J. CLIN. MICROBIOL. An outbrek of tuberculosis cused by multiple-drug resistnt tubercle bcilli mong ptients with HIV infection. Ann. Intern. Med. 117: Godfrey-Fussett, P., P. R. Mortimer, P. A. Jenkins, nd N. G. Stoker Evidence of trnsmission of tuberculosis by DNA fingerprinting. Br. Med. J. 305: Groenen, P. Unpublished dt. 11. Hermns, P. W. M., D. vn Soolingen, E. M. Bik, P. E. W. de Hs, J. W. Dle, nd J. D. A. vn Embden The insertion element IS987 from Mycobcterium bovis BCG is locted in hot-spot integrtion region for insertion elements in Mycobcterium tuberculosis complex strins. Infect. Immun. 59: Hermns, P. W. M., D. vn Soolingen, J. W. Dle, A. R. Schuitem, R. A. McAdm, D. Ctty, nd J. D. A. vn Embden Insertion element IS986 from Mycobctenium tuberculosis: useful tool for dignosis nd epidemiology of tuberculosis. J. Clin. Microbiol. 28: Hermns, P. W. M., D. vn Soolingen, nd J. D. A. vn Embden Chrcteriztion of mjor polymorphic tndem repet in Mycobcterium tuberculosis nd its potentil use in the epidemiology of Mycobcterium knssii nd Mycobcterium gordone. J. Bcteriol. 174: Mzurek, G. H., M. D. Cve, K. D. Eisench, R. J. Wllce, Jr., J. H. Btes, nd J. T. Crwford Chromosoml DNA fingerprint ptterns produced with IS6110 s strin-specific mrkers for epidemiologic study of tuberculosis. J. Clin. Microbiol. 29: McAdm, R. A., P. W. M. Hermns, D. vn Soolingen, Z. F. Zinuddin, D. Ctty, J. D. A. vn Embden, nd J. W. Dle Chrcteriztion of Mycobcterium tuberculosis insertion sequence belonging to the IS3 fmily. Mol. Microbiol. 4: Mendiol, M. V., C. Mrtin, I. Otl, nd B. Gicquel Anlysis of regions responsible for IS6110 RFLP in single Mycobcterium tuberculosis strin. Res. Microbiol. 143: Otl, I., C. Mrtin, V. Vincent-Levy-Frebult, D. Thierry, nd B. Gicquel Restriction frgment length polymorphism nlysis using IS6110 s n epidemiologicl mrker in tuberculosis. J. Clin. Microbiol. 29: Rstogi, N., B. C. Ross, B. Dwyer, K. S. Goh, S. Clvel-Seres, V. Jentils, nd P. Cruud Emergence during unsuccessful chemotherpy of multiple drug resistnce in strin of Mycobcterium tuberculosis. Eur. J. Clin. Microbiol. Infect. Dis. 11: Rogll, T., J. Wolters, T. Flohr, nd E. C. Boettger Towrds phylogeny nd definition of species t the moleculr level within the genus Mycobcterium. Int. J. Syst. Bct. 40: Ross, C., K. Rios, K. Jckson, nd B. Dwyer Moleculr cloning of highly repeted element from Mycobcterium tuberculosis nd its use s n epidemiologicl tool. J. Clin. Microbiol. 30: Schoningh, R., C. P. H. J. VerstUnen, S. Kuiper, nd A. H. J. Kollk Enzyme immunossy for identifiction of hetkilled mycobcteri belonging to the Mycobcterium tuberculosis nd Mycobcterium vium complexes nd derived from erly cultures. J. Clin. Microbiol. 28: Smll, P. Personl communiction. 22. Smll, P. M., R. W. Schfer, P. C. Hopewell, S. P. Singh, M. J. Murphy, E. Desmond, M. F. Sierr, nd G. K. Schoolnilk Submitted for publiction. 23. Smith, D. W Diseses in guine pigs, p In G. P. Kubic nd L. G. Wyne (ed.), The mycobcteri: sourcebook. Prt B. Mrcel Dekker, Inc., New York. 24. Suzuki, Y., A. Ngt, Y. Ono, nd T. Ymd Complete nucleotide sequence of the 16S rrna gene of Mycobcterium bovis BCG. J. Bcteriol. 170: Thierry, D., A. Brisson-Noel, V. Vincent-LUvy-Fr6bult, S. Nguyen, J. Guesdon, nd B. Gicquel Chrcteriztion of Mycobcterium tuberculosis insertion sequence, IS6110, nd its ppliction in dignosis. J. Clin. Microbiol. 28:

9 VOL. 31, 1993 M. TUBERCULOSIS DIFFERENTIATION AND EPIDEMIOLOGY Tisdll, P. A., D. R. Deyoung, G. D. Roberts, nd J. P. Anhlt Identifiction of clinicl isoltes of mycobcteri with gs-liquid chromtogrphy: 10-month follow-up study. J. Clin. Microbiol. 16: Vn Embden, J. D. A., M. D. Cve, J. T. Crwford, J. W. Dle, K. D. Eisench, B. Gicquel, P. W. M. Hermns, C. Mrtin, R. Mcdm, T. M. Shinnick, nd P. M. Smll Strin identifiction of Mycobctenium tuberculosis by DNA fingerprinting: recommendtions for stndrdized methodology. J. Clin. Microbiol. 31: Vn Embden, J. D. A., D. vn Soolingen, P. M. Smll, nd P. W. M. Hermns Genetic mrkers for the epidemiology of tuberculosis. Res. Microbiol. 143: Vn Soolingen, D. Unpublished dt. 29. Vn Soolingen, D., P. W. M. Hermns, P. E. W. de Hs, D. R. Soll, nd J. D. A. vn Embden The occurrence nd stbility of insertion sequences in Mycobcterium tuberculosis complex strins; evlution of IS-dependent DNA polymorphism s tool in the epidemiology of tuberculosis. J. Clin. Microbiol. 29: Vn Soolingen, D., P. W. M. Hermns, P. E. W. de Hs, nd J. D. A. vn Embden Insertion element IS1081-ssocited restriction frgment length polymorphism in Mycobcterium tuberculosis complex spcies: relible tool for recognizing Mycobcterium bovis BCG. J. Clin. Microbiol. 30: Wyne, L. G Mycobcteril specition, p In G. P. Kubic nd L. G. Wyne (ed.), The mycobcteri: sourcebook. Prt A. Mrcel Dekker, Inc., New York. 32. Zinuddin, Z. F., nd J. W. Dle Polymorphic repetitive DNA sequences in Mycobcterium tuberculosis detected with gene probe from Mycobcterium fortuitum plsmid. J. Gen. Microbiol. 135: Downloded from on Mrch 25, 2019 by guest