Insertion element IS1081 associated restriction fragment length polymorphisms in Mycobacterium tuberculosis complex species: a reliable tool for recognizing Mycobacterium bovis BCG

0095-1137/92/071772-06$02.00/0

Copyright C 1992, AmericanSociety for Microbiology

Insertion Element IS1081-Associated

Restriction

Fragment

Length

Polymorphisms

in

Mycobacterium

tuberculosis

Complex Species:

a

Reliable

Tool for

Recognizing

Mycobacterium

bovis

BCG

DICKVANSOOLINGEN,' PETER W. M. HERMANS,2PETRA E. W. DEHAAS,' ANDJAN D. A.VAN EMBDEN2*

Laboratory of Bacteriology' and UnitofMolecularMicrobiology,2NationalInstituteofPublic Healthand EnvironmentalProtection, P.O. Box1, 3720 BABilthoven, TheNetherlands

Received 27 January 1992/Accepted10April1992

Recently,the insertion elementIS1081fromMycobacteriumboviswasidentified.In thisstudy,theusefulness ofIS1081 intheepidemiologyof tuberculosis wasinvestigated.The hostrangeof this insertion sequencewas

found to be restrictedexclusivelytothegroupofMycobacteriumtuberculosiscomplex bacteria,whereasnone

of the 10 mycobacterial species which do not belong to the M. tuberculosis complex contained IS1081-homologousDNA. All 99 M. tuberculosiscomplexstrainsinvestigatedcarried fiveorsixcopiesofIS1081, and

verylimited IS1081-associated restrictionfragmentlength polymorphismswere observedamongthestrains. Seven differentIS1081-containingbandsweredistinguishedin eachstrain,andthepatternsdifferedonlyinone ortwoinsertion sequence-containingbands. Thebanding patternof M. bovis BCG differedin thepresenceof

a8.0-kbIS1081-containingPvuIIfragmentwhichwasabsent from all other M. tuberculosiscomplexstrains.

Mycobacterium tuberculosis

complex

bacteria have

re-centlybeen foundtocontainaninsertion sequence,IS6110, which belongs tothe IS3familyofenterobacterial insertion sequence (IS) elements. The sequences of three

copies

of these IS elements, IS6110and IS986 from M. tuberculosis and IS987 from Mycobactenum bovis

BCG,

have been described previously (7,

12,

16), and all were found to be virtually identical(7).

The host range ofthis insertion element is limited to the species of M. tuberculosis, M. bovis,

Mycobactenum

afti-canum,andMycobacteriummicroti; and among hundreds of M. tuberculosis complex isolates that have been investi-gated,none wasfoundtobe devoid of this IS element(8, 18). Therefore, this ISelement is of considerable practicalvalue as aDNA targetin thepolymerasechain reaction(PCR)for detection of tuberculosispathogensinclinicalspecimens (2, 8, 15).

Furthermore, the variabilities in copy number and chro-mosomal location have been shown tobe useful for geneti-cally distinguishing M. tuberculosis strains by means of restriction fragment length polymorphism (RFLP) (3, 8, 13, 18, 20). The number of IS copies per chromosome differs greatly among different isolates and certain species such as M.

africanum

and M. tuberculosis, which tend to harbor more copies than do other species like M. bovis (18). The high degree of polymorphism of IS-containing restriction fragments among M. tuberculosis complex isolates has been found tobe extremely useful forepidemiological studies (8, 18).

In an attempt to identify DNA sequences which would enable thedifferentiation of M. tuberculosis from M. bovis, Collins and Stephens (4) have recently cloned a DNA sequencefrom M. bovis; the sequence was found to contain aninsertion element that greatly differs from the IS3family. Thisinsertion element, IS1081, is 1,324 bp in size, and the

*Correspondingauthor.

putative transposase bears some resemblance to that of IS256 ofStaphylococcus aureus (4).

Inthis study,we investigated the host range of insertion element IS1081 among the variouspathogenic and nonpath-ogenic mycobacterial species and the genetic polymorphism associated with theputativetransposableelement. We show that this IS element isexclusivelypresentin M. tuberculosis complex species and that IS1081 can be used to reliably differentiate M. bovis BCG from other M. tuberculosis complexbacteria.

MATERIALSANDMETHODS

Mycobacterial strains. The mycobacterial strains used in thisstudyarelisted in Table 1.Culturingof themycobacteria wasperformed asdescribedpreviously (18).

DNAtechniques.Purificationof chromosomal mycobacte-rial DNA and hybridization techniques were done as de-scribedinourpreviousreport(18).Unless otherwisestated, therestriction enzyme PvuIIwasusedtodigest mycobacte-rial DNA prior to electrophoresis. The IS986-specific 245-bp PCR-amplified fragment and the 386-bpXhoI-BamHI frag-mentofpRP5000were used asDNAprobes for hybridiza-tion (8). The IS1081-specific DNA probe of 300 bp was amplified by PCR byusing the oligonucleotides 1081a (5'-TCGCGTGATCC1TCG) and 1081b (5'-CGCAGCTTGGG GATCGCGAC), which are based on the inverted repeat sequence at positions 333 to 347 and the sequence at positions613to632 of the IS1081 sequence, respectively (4). DNA amplification by PCR was performed as reported previously(8). For hybridization experiments, the enhanced chemiluminescence genedetection system was used (Amer-sham International plc.).

Biochemical tests and growth characteristics. Bacteriologi-caldetermination of M. bovis and M. bovis BCG was based oncolony morphology, susceptibility to thiophene-2-carbox-ylic acid hydrazide, growth on L6wenstein-Jensen medium in the presence or absence of pyruvate, nitrate reductase

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TABLE 1. Bacterial strains used in this study

Bacterial strain Species Property or origin Source or reference

108-111, 114, 116, 117, Mycobacterium tuberculosis Clinical isolates Our laboratory 119-126, 286-292

324-333 Mycobacterium tuberculosis Human clinical isolates (Ruwanda) F. Portaelsa

334-335 Mycobacteriumtuberculosis Human clinical isolates (Central F. Portaelsa

African Republic)

336-343 Mycobacterium tuberculosis Human clinical isolates (Burundi) F. Portaelsa

38,153 Mycobacteriumafricanum Clinical isolates Our laboratory

40, 41, 44, 154, 168-170, Mycobacterium bovis Human clinical isolates Our laboratory 296-301, 379-386

369-378 Mycobacterium bovis Bovineclinicalisolates J. van derGiessenb

45, 105, 150-152,384 Mycobacterium bovis BCG Clinical isolates Our laboratory

102 Mycobacterium bovis BCG Vaccine strain OrganonTeknikac

103 Mycobacterium bovis BCG Vaccine strain ArmandFrappierd

44 Mycobacterium bovisBCG Vaccinestrain Ourlaboratory

46 Mycobacteriummicroti F. Portaelsa

158 Mycobacteriumasiaticum Ourlaboratory

49 Mycobacteriumavium Ourlaboratory

156 Mycobacteriumflavescens Ourlaboratory

155 Mycobacterium scrofulaceum

159 Mycobacteriumgordonae Ourlaboratory

160 Mycobacterium kansasii Ourlaboratory

157 Mycobacterium malmoense Ourlaboratory

163 Mycobacterium terrae Ourlaboratory

162 Mycobacterium chitae ATCC 25805e

161 Mycobacteniumintracellulare ATCC19422e

Escherichia coliK-12 Ourlaboratory

Bordetella pertussis Wellcome 28 14

aPrinceLeopold InstituteofTropicalMedicine, Antwerp, Belgium.

^Veterinary Department, UniversityofUtrecht, Utrecht,TheNetherlands. Organon Teknika N.V.,Veedijk58-2300Turnhout,Belgium.

dInstitut ArmandFrappier, Laval, Quebec,Canada.

eAmericanType CultureCollection, Rockville, Md.

activity, and the presence of serpentines of bacteria by microscopic analysis(17, 19).

RESULTS

Host rangeofIS1081amongvariousmycobacterial species.

Toinvestigatethe hostrangeofIS1081 amongspecies of the genus Mycobacterium, NruI-digested chromosomal DNA fromvarious mycobacterial specieswas analyzed by South-ern blotting by using the 300-bp IS1081 probe. As shown in

Fig. 1, onlystrains from the M. tuberculosiscomplexgroup,

M. tuberculosis, M. africanum, M. bovis BCG, and M. microti, hybridizedwith theIS1081 probe. Incontrast,none

of the other bacterial species tested, Mycobacterium

fla-vescens,Mycobacterium malmoense, Mycobacterium

asiat-icum, Mycobacterium avium, Mycobacterium gordonae, Mycobacterium kansasii, Mycobacterium intracellulare, Mycobacterium chitae, Mycobacterium terrae,Escherichia

coli, and Bordetellapertussis, hybridized with this probe. We conclude that, similar to IS986, the insertion element

IS1081 is specifically present only in the M. tuberculosis

complex species.

IS1081-associatedRFLP in M. tuberculosiscomplexstrains.

All M. tuberculosis complex species, including two M.

tuberculosisstrains, twoM.bovisstrains,two M. africanum strains, andM. microti one strain, showedavirtually

iden-tical pattern ofsixIS1081-hybridizingNruI fragments, sug-gesting that the IS1081 copies have an identical

chromo-somal location in these strains of the M. tuberculosis

complex. However, by usingtherestrictionenzymePvuIIin

Southern blot experiments, IS1081-associated RFLPs were

observedamongthe M. tuberculosiscomplexstrains.Figure

1 2 3 4 5 6 7 8 9 1 0 I1 70 13 74!; 1 13 1 21 .7. 2.13

-4

--1 4

-0.6

FIG. 1. OccurrenceofIS1081 invarious

mycobacterial species

determined

by

Southern blot

analysis

of

NruI-digested

chromo-somal DNA from different

species

hybridized

with the

300-bp

PCR

fragment

ofIS1081. Lanes: 1,M. tuberculosis 111; 2,M. tubercu-losis112; 3,M.

africanum

38; 4,M.

africanum

153; 5,M.bovis154; 6,M.bovis41; 7,M. bovis BCG102; 8,M. bovis BCG103; 9,M. microti 46; 10, M.

scrofulaceum;

11, M.

flavescens

156; 12, M. malmoense 157; 13,M. asiaticum 158; 14, M. avium 49; 15, M.

gordonae

159;16,M.kansasii160; 17,M.intracellulare161;18,M.

chitae 162; 19,M. terrae163; 20,B.

pertussis

Wellcome28;21,E. coli K-12; 22, noDNA. Numberson the left indicate the sizes of standardDNA

fragments (in

kilobase

pairs).

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A A A A A A A A A B B A A A A A A A A A B

a 1 5 6 H 9 1i)l 1 2 I1 i I f; 111 I 2 2 I...

'1 4 ..

.,rz _

4.4

-OO- W - WN - -m - am amm

=-S" 5 -- - -m -0 -m - - -0 -lo - -0*W

-2 3

-27.(1

-A A AA A A A A A B B AA A A A A A A A B

b 2 I, I,1) ; '@

li.

1.

111111

1.1i:';1113.ll;.2

I

t',... _z

- _ _

z~~~~ ~ ~ ~

__ = __ -

u

--~

_--

----m3S

-m

- - _ _

FIG. 2. ComparisonofIS1081- andIS986-associated RFLPs among Dutch M. tuberculosis isolates.PvuII-digested chromosomalDNA was used inSouthern blothybridizationwith the300-bp probe of IS1081 (a) and the 386-bp probe of IS986(b).Lanes 1 to21, strains 286,287, 288,289,290, 291, 108, 121, 109, 122, 111, 126, 292, 114, 124, 123, 119, 120, 116, 125, and 117,respectively. Numbers on the left indicate sizes of standard DNAfragments(inkilobasepairs). The capital letters denote the IS1081PvuII fingerprinttype.

2a shows the results of Southern blot analysis of chromo-somal DNAs from 21 clinical isolates of M. tuberculosis originating from a Dutch regional public health laboratory. Each strain showed six hybridizing bands; because IS1081 does notcontain aPvuII site, these data indicate that these strains carry sixcopies of the insertion element IS1081. The degree of polymorphism in the banding patterns was very limited. All patterns except those from three isolates were identical(typeA),and the three patterns that were different from those of themajorityof isolates (Fig. 2a, lanes 10, 11, and 21) were identical (type B). The two banding patterns differed only in theposition of oneIS1081-containingband, whereas the other five IS copies were presenton the same PvuII fragments.

To compare the IS1081 banding patterns with the DNA fingerprintsobtainedwith IS986, thesamefilterwasused for hybridization with labeled IS986 DNA (Fig. 2b). As ex-pected,theIS986bandingpatternsdifferedgreatlyfrom the IS1081 fingerprints, in that the IS986-containing banding patterns were very polymorphic. All IS986 fingerprints in Fig. 2bdiffered from each other.

C A A A C A D A A E A A A A A D 0 A A A

a 2 .1 43, ( / 8 9 11 1111.21III1 131(i I I)18 19121 21

.) .1

--_ ---___----___

2.

--6i. 6

--4 .--4t

am - - - -__1__ - _ _ _ _

As describedpreviously (18), strains from Africaappearto

be less polymorphic with regard to the IS986-containing restrictionfragments compared with strains of Dutchorigin. Therefore, DNA fingerprints of these African strains were

madeby using IS1081 as aprobe (Fig. 3). Again, little RFLP was observed; 13 of the 20 strains tested displayed an identical fingerprint, type A, which was also the

predomi-nant fingerprint type amongthe Dutch strains. The remain-ingsevenstrains displayed three differentpatterns, typesC, D,and E(Fig. 3a),andagain, each of these strains sharedat

least five bands with the type A strains. All strains except those of type D were found to carry six copies ofIS1081; type D strains contained five IS1081 elements. As shown in Fig.3b, a much greater RFLP was observed when the IS986 DNA probe was used. The three strains with IS1081banding pattern D showed littlepolymorphism in the IS986-contain-ing banding patterns, indicating that these three strains belong to aclosely related group.

Twenty-two M. bovis strains (8 isolated from cows, 2 isolated from llamas, 11 isolated from humans, and 1 M. bovis BCG vaccine strain), were analyzed by DNA

finger-C A A A C A D A A E A A A A A D A A A

b .1 *, ', t 3 91; 11 1.3 1114115 111 11 111 ]') 21} .31

1-~~ ~ ~ ~

- --mm -i

.1- in * m m

~~~?,

-FIG. 3. Comparison ofIS1081-andIS986-associated RFLPs among African M. tuberculosis isolates. Lanes 1 to 20, strains 324 to343,

respectively; lanes 21, no DNA. Southern blotting and hybridization were performed as described in the legend to Fig. 2. Numbers on the leftindicatesizes ofstandard DNA fragments (in kilobase pairs). The capital letters denote the IS1081PvuIIfingerprint type.

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AA A A A A A A A A A A A A A A A C F A G G

a 2 4 f1 21112li 1 1111113if21, ',.

4*4 - I'X aI t IS4 a - r' __

,. ~X -..-. "-b---. ._n_.

_-~~~~M.* I - e- - on OM - - = _ _

A A A A A A A A A A A A A A A A A C F A G G

b 1 2 3 4 5 6 '7 8 9 011 12 13 14 15 1f 17 1819 21 21 22

.-.6

-....

4.4

----~~~~~~~~~~~~~~~~~~~~~~~~2

2.

-2.0

-C]

--OJ.6 -l}.f6

-U

_ _ - - -_a_

a__

__

FIG. 4. DNAfingerprinting of various M. bovis strains of different clinical origins by using the 300-bp probe ofIS1081(a)and the 245-bp probeIS986 (b). M. bovis strains originated from animals (lanes 1 to 10) and humans (lanes 11 to 20). Lanes 21 and 22, the hybridization patternof theDutch M. bovis BCG vaccine strain 44 and the M. bovis BCG human clinical isolate 383, respectively. Strains 369 to 382 (lanes 1 to14,respectively)and strains 296, 384, 171, 168, 40, and 41 (lanes 15 to 20, respectively) were analyzed by Southern blotting. Hybridization wasperformed as described in the legend to Fig. 2. Numbers on the left indicate sizes of standard DNA fragments (in kilobase pairs). The capital letters denote the IS1081PvuIIfingerprint type.

printingby using IS1081 and IS986 DNAs as probes. Like the M. tuberculosis isolates, the M. bovis isolates displayed verylimited IS1081-associated RFLPs (Fig. 4a and Table 2), incontrast to thefingerprints generated when labeled IS986 DNA as a probe(Fig. 4b). Eighteen of 22 strains tested were of type A, thepredominant type among the M. tuberculosis isolates. All bovine M. bovis strains were of this type; and three other patterns, types C, F, and G, were observed among the human M. bovis strains. Similar to previous observations(18), these strains showed much more polymor-phism when the same filters were hybridized with labeled IS986 DNA; among the 21 isolates tested, 13 different IS986-containing restriction fragment patterns were found. Two strains displayed the banding pattern G, and one of these strains was a known M. bovisBCGvaccine strain (Fig. 4, lanes21). Biochemical redetermination revealed that the other G-type strain(Fig. 4, lanes 22), a human isolate, was misidentified as an M. bovis strain. On the basis of equal growth on Lowenstein-Jensen medium with and without pyruvate, a rough colony morphology, inhibition by thiophene, and the presenceofacordfactor,this strainwas determined tobe M. bovis BCG.

Differentiation of M. bovis BCG from M. bovis by DNA fingerprinting. The observation that the twoM. bovis BCG strains differed in their IS1081-containing PvuII banding patterns could suggest that DNA fingerprinting might be useful for distinguishing M. bovis BCG strains from wild-type M. bovis strains. Therefore, 26 M. bovis BCG strains were analyzed by DNA fingerprinting, and 14 human M.

TABLE 2. Distribution ofIS1081 DNAfingerprinttypesamong M. tuberculosis complexstrains

No. of No. of strains withfollowing

Species strains DNA fingerprint type:

tested A B C D E F G

Mycobacterium tuberculosis 41 32 3 2 3 1 0 0

Mycobacterium bovis 32 29 0 2 0 0 1 0

Mycobactenium bovisBCG 26 0 0 0 0 0 0 26

bovisisolates were tested for comparison. Figure 5 illustrates the results. All 26 M. bovis BCG strains contained the same DNAfingerprint, typeG, asdescribed above for the two M. bovis BCG strains. Noneof the M. bovis isolates were of this fingerprint type. All differed in the absence of the 8.0-kb IS1081-containing PvuII fragment, which was invariably found inM. bovis BCG(Fig. 4). The distributions of the seven differentIS1081 DNAfingerprint types among the M. tuber-culosiscomplex strains are summarized in Table 2.

A A A A C A A A A A A A G G G G G G G

I .' -l1 fU / f lIi II I.111 4 1 i1Il 1811)

4.4

- 1.6-2). G

-FIG. 5. RFLPtypingof various strains ofM.bovisand M. bovis BCG.The300-bpDNAprobeofIS1081wasused in Southern blot hybridization.Lanes 1to12,M.bovis169, 170, 168,154,171, 297,

298,385, 386, 299, 300, and 301, respectively; lanes 13 to 19,M. bovis BCG 150, 45, 103, 102, 152, 151, and150, respectively. All strains except the M. bovis BCG vaccine strains 102 and 103were isolated from humans.Experimentalprocedureswereperformedas described in thelegendtoFig.2.Numbersonthe left indicate sizes ofstandard DNAfragments (inkilobase pairs).Thecapital letters denote theIS1081PvuII fingerprinttype.

* z

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DISCUSSION

Results of this

study

indicate that the IS1081-associated DNA

polymorphism

of

IS-containing

restriction

fragments

is verylimited. All 99 M. tuberculosis

complex

strains

inves-tigated

werefound tocontain fiveorsix

IS1081

copies.

The

predominant fingerprint

pattern among M. tuberculosis and M. bovis strains was

pattern

A

(61

of 99 strains

tested),

which consisted of sixPvuII

fragments

of

6.2, 4.4, 4.3, 3.9,

3.6,

and 1.8

kb,

respectively.

This lack of

polymorphism

indicates that IS1081 transposesvery

rarely,

or

perhaps

not at

all,

in these

mycobacteria.

The very limited RFLPs associated with

IS1081

might

also be caused

by

some

vari-ability

in the chromosomal DNA

flanking

the insertion element.

The apparent extreme

stability

of

IS1081

is in

sharp

contrast tothat of the IS3-likeelement IS986 in M. tubercu-losis

complex

strains,

the

only

otherknown IS in this group of

pathogenic mycobacteria.

The latter element appears to

transpose

relatively frequently,

because

virtually

every

non-epidemiologically

related isolate has been foundtocarrythe IS element on different restriction

fragments (3,

8, 13, 18,

20).

Fromall strains

investigated

in this

study,

wealso made

IS986

fingerprints,

and as

expected,

virtually

all strains

differed in their

IS986-containing banding

patterns,

confirm-ing

the

previous

observationsonthe relative

high

frequency

of

transposition

of the IS3-like insertion element.M. tuber-culosis isolates with

closely

related

IS986

fingerprints

were

found to have identical

IS1081

fingerprints

if

they

also exhibiteda

banding

different from those of the

predominant

type

Astrains.

M. bovis BCG differed from all the other M. tuberculosis

complex

strains

investigated

in the presence of a 8.0-kb

IS1081-containing

PvuII

fragment.

In this

study,

26M.bovis BCG strains were

investigated;

20were isolated from hu-mans and 6 were vaccine strains.

Although

the

majority

of

the M. bovis BCG strains carry a

single

IS3-like

element,

IS987,

at a

unique

chromosomal

position (7),

three vaccine

strains have been shown to contain twoIS

copies (6).

The latter strains are the vaccine strains used in

Russia, Japan,

and

Brazil;

and thesestrainswerealsoincluded inour

study.

The latter three strains also showed the

BCG-specific

PvuII IS1081

banding

pattern

G,

in which one IS1081 copy is locatedon a 8.0-kb PvuII

fragment.

These data suggest that

this chromosomal DNA

fragment

of M. bovis BCG differs in

a

unique

way from all other M. tuberculosis

complex

strains.

At

present,

it isnotclear whether this difference is duetothe insertionof

IS1081

intoadifferent

region

ofthe chromosome orto adifference inthe chromosomal

region

thatflanksone

ofthe six IS1081

copies

in M. bovis BCG.

M. bovis BCG is

notoriously

difficulttodifferentiatefrom M. bovis

by

classicalbiochemical reactionsor

growth

char-acteristics

(19).

Forthis reason,

analysis

of

phage

suscepti-bility

or

pathogenicity

to animals is used to

distinguish

M. bovis BCG from

wild-type

M. bovis strains

(5,

9). However, these methodsare

time-consuming

anddifficult toperform. This

study

showed that the

unique

BCG patterns of

IS1081-containing

restriction

fragments

areof greatuseinthetyping of M. bovis BCG. The

possibility

ofeasily differentiatingM. bovis BCG from

pathogenic

mycobacteria will be

increas-ingly

important

in

light

ofthe

increasing

numberofhumans with human

immunodeficiency

virus infections. As myco-bacterial infections reactivate

during

human

immunodefi-ciency

virus-induced

immunosuppression,

dormantM. bovis BCG bacteria

might

reactivate and cause a disease that resemblestuberculosis.

Recently,

suchacasewasconfirmed

in a human immunodeficiency virus-infected individual who displayed a reactivation of M. bovis BCG 30 years after vaccination (1). Preliminary studies suggest that patients with AIDSwhoare infectedwith M. tuberculosis aremore infectious than nonimmunosuppressed individuals and, therefore, might disseminate pathogenic mycobacteriamore

rapidly in the community. To establish the hazard relatedto such increased infectivity, it will be increasingly important tohave asimple tool todifferentiatepathogenic M. tubercu-losis complex bacteria from the attenuated strain M. bovis BCG.

M. bovis BCG is increasingly used as aneffective immu-nostimulatory agent to prevent relapses of urinary bladder carcinoma (11). However, complications of this treatment canoccur, resulting in local or disseminated infections that causegranulomatouspathology in variousorgansortissues (10). Correct identification of M. bovis BCG is of great importance for distinguishing M. bovis infections and M. bovis BCGtreatmentcomplications.

At present, the reason for the exclusive presence ofthe 8.0-kb IS1081-containing restriction fragment in M. bovis BCG is unknown. However, it is tempting to speculate on thepossibilitythat thisunique genetic propertyis related to the nonvirulence of this mycobacterium, perhaps by a ge-netic rearrangement induced by IS1081 in a chromosomal region, which is essential forpathogenicity.

ACKNOWLEDGMENTS

We thank J. G. Baas and J. E. M. Pijnenburg for beneficial technical assistance and J.vander Giessen forprovidinguswithM. bovis strains. A. G. Vilasi is acknowledgedfor perfectsecretarial

assistance,andwethank thePhotography Department forexcellent service.

This study received financial support from the Program for VaccineDevelopment,WorldHealthOrganization,and Science and Technology Development, European Community.

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