Identification of sequence types among the M nontypeable group A streptococci

Copyright X 1992,American Society for Microbiology

Identification of

Sequence Types among the M-Nontypeable

Group

A

Streptococci

WENDYA. RELF,1DIANAR.MARTIN,2ANDKADABA S. SRIPRAKASHl*

MenziesSchool of Health Research, PO Box 41096, Casuarina,

Australia,

1 and NewZealand

CommunicableDisease Centre, PO Box 50348, Porirua, NewZealand2

Received9July 1992/Accepted 1September 1992

Streptococcal diseases,

namely,

acuteglomerulonephritis and acute rheumatic fever, are common features in theaboriginal population of the Northern Territoryof Australia. We examined the groupAstreptococcal M

types identified during various surveys conducted since 1987. Streptococci were

predominantly

isolated from skin infections. A high proportion of the isolates could not be serotyped by conventional means and were designated M nontypeable (MNT). M-specific DNA sequences from the MNT isolates were examined, and sequencetypeswere proposed for the classification of MNTs. This allowed a more preciseestimate of theM

types presentin apopulation study.

Thereis a high prevalence of group A streptococci (GAS) thatcauseinfections among Australian aboriginal children, and the incidence of acute rheumatic fever remains high in

aboriginal communities (3). Epidemics ofacute

glomerulo-nephritisoccurredinaboriginal communities in the Northern

Territory of Australia in 1965, 1969, and 1973, but no organisms were specifically identified. An outbreak of

ne-phritisin 1980wasattributedtostreptococciof type M55(6),

and in late 1987, Ml and M-nontypeable (MNT) isolates were implicated in another acute glomerulonephritis epi-demic. Our understanding of the epidemiology of strepto-coccal diseasein the NorthernTerritoryhas beenrestricted

by theinabilityof theavailabletyping antiseratoidentifythe M typesofalarge proportionof isolates.

The serology of GAS is based on recognition of the M proteinonthe surface of isolates.Althoughmorethan 80 M types are recognized internationally, a number of strepto-coccal isolates, particularly from skin infections, are

fre-quently MNTbecause ofalack ofreactivity with available

reference M antisera rather than a lack of M protein (16). Distinctionof MNTisolates canbe increased if the supple-mentaryopacityfactor (OF)inhibition testis used (17). OF isanapoproteinase (25) expressed by allmembers of certain M types, manyofwhichcauseskin infections. The

specific-ity of OF parallels that of Mprotein (7),and therefore, OF

typinginfers therecognition ofsomeM-proteintypes (17).

Inthestudydescribed here,weinvestigatedthe M types of GAS isolated inthe NorthernTerritoryofAustralia from 1987to1991. Alargeproportionof isolateswereMNT.We also describe the use of DNA sequencing both for the validation of the M type obtained serologically and to

characterize isolates that were serologically nontypeable. The designation ofa sequence type (ST) was assigned for distinctMNTisolates.

MATERIALSANDMETHODS

Bacterial isolates andserotyping. Epidemiological surveys of streptococcal infections in the Northern Territory of Australia were conducted from 1987to 1991.A total of 82 isolates associated with infection and other residential

or-ganismswereexamined in thestudy.Swabs from thethroat,

* Correspondingauthor.

moist orimpetiginous lesions,and/or the axillae takenfrom children residing in four distinct aboriginal communities in theNorthern Territory provided67isolates.Fifteen isolates

ofGAS werefrom patientswith sporadic invasive

strepto-coccal disease and other complications identified at Royal

Darwin HospitalandAliceSprings Hospital.

Themethods for isolation andidentificationofGAS have been described previously (26). Briefly, the bacteria were

plated onto blood agarcontaining colistin (10 mg/liter) and

oxolinic acid (5 mg/liter). Streptococci were grouped by

using commercial reagents (Karo-bio Phadebact;

Pharma-cia). Streptococcal isolateswere characterized by M type,

T-agglutination pattern, and OF serotyping by standard

methods (15). Antisera for M typing, T typing, and OF

typingwereprepared inthe StreptococcalReference Labo-ratory atthe New Zealand Communicable Disease Centre

againstthe International Set of Reference Type Strains. M

typingwas performed bythe Ouchterlony double diffusion method of Rotta(24). T-agglutination patternswereobtained bythe method ofGriffith(8),andOFserotypingwasdoneby

the method of Maxtedetal.(17).AntiseratothefollowingM

proteinswereused: 1, 3, 3R, 5, 6, 12, 14, 18, 24, 26, 29, 41, 52, 53, 55, 57,and 80. OF antisera for types2, 4, 13, 22,25,

28, 49, 58, 59, 60, 61, 63, 66, 68, 73, 75, 77, 81, PT180, NZ1437, PT1658, Tr2407, Tr2612, PT2841, PT3875, and NZ5118wereused forOF-positiveorganisms.

PCR and DNAsequencing.StreptococcalDNAsfrom25of the 82 isolateswereisolated and

amplified

withpolymerase

chain reaction

(PCR)

primer sets on the basis of the

con-served leader and C-terminal regions of the M protein of GAS asdescribedpreviously

(22).

The PCRproductswere

purifiedfromexcessoligonucleotide primers byisopropanol precipitation. High-efficiency cloningwas achieved (13) by

self-concatemerization of the PCR product with T4 DNA ligase(Promega)anddigestingwith BamHI andPstI

restric-tion enzymes (Promega) before ligating into the vector

pUC19. Following

transformation,

dideoxysequencingwas

performed on plasmidDNAs from three clones containing streptococcalinserts. The DNAanalysisprogramMatch(27)

was used to compare the sequences of the isolates with a

data base of nucleotide sequences corresponding to the N-terminal ends of various M and M-like proteins, as fol-lows: Ml (10);ML2, M3 (20); M3R(21); M5(18); M6(12);

M12 (23); M13, M14, M17 (21); M18, M19 (20); M22

(21);

3190

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TABLE 1. GAS isolates collected in theNorthern Territory of Australia since 1987

No. ofisolates

Community' Yearof Mtype Designation totalno.of ST

isolates

A 1987 Ml 88/32, 88/35 2/2

MNT 88/23, 88/26,88/28,88/30, 88/31, 88/33, 88/34 7/7 STDONALD

MNT 88/25 1/1 ST88/25

MNT 88/24, 88/27,88/29 0/3

A 1989 MNT 89/372, 89/373,89/374, 89/375, 89/376,89/377, 89/378, 89/379, 0/10 89/380,89/381

B 1989 MNT 89/548 1/1 ST548

MNT 89/550 1/1 ST550

MNT 89/544, 89/545, 89/546, 89/547,89/549, 89/551 0/6

C 1990 M4 90/27 1/1

M57 90/31 ,

90/80b

2/2

MNT 90/29, 90/84, 90/85,90/87 4/4 ST90/85

MNT 90/86 1/1 ST88/25

D 1991 M52 BSD34 0/1

M53/80 NS27b,BSA54, BSB54,BSE54, BSB12, BSB36, BSB60 0/7

M55 NS38b,BSE52, BSB63, BSA75 0/4

M57 BSB38,BSA57 0/2

M61/PT3875 BSB34,BSB10, BSA67 0/3

M63 BSA39,BSB39, BSB41, BSA41 0/4

M80 BSB75 0/1

NZ1437 BSA52 0/1

MNT BSD39,BSA12, BSA63,BSA10,BSA15 0/5

Hospital 1990 M6 NS4 0/1

M12 NS9 1/1

M53 NS10b, NS11, NS13 2/3

M61/PT3875 NS12 0/1

M80 NS5 0/1

MNT NS1 1/1 STNS1

MNT 1293 1/1 ST1293

MNT NS14 1/1 STNS14X

MNT 1275, NS2, NS6, NS7, NS8 0/5

aA, B,C, andD aredifferentcommunities in theNorthernTerritoryofAustralia.HospitalisolatesarefromRoyalDarwinHospitaland AliceSprings Hospital.

bThese isolatesarefrompatientswithacuteglomerulonephritis.

M24(19);M30(21);M49(9); M52, M53,M55(21);M57(14); M60, M80(21); Arp4 (5); EnnX(9); andFcRA76(11).

Nucleotidesequenceaccession numbers. TheGenBank ac-cession numbers for thefollowingS.pyogenesstrainsare as follows: STDONALD (243 bp), L05017; ST88/25 (219 bp), L05018; ST548 (222 bp), L05019; ST550 (177 bp), L05020; ST90/85 (243 bp), L05021;STNS1(144 bp), L05022; ST1293

(142 bp), L05023; STNS14X(177 bp), L05024. RESULTS

Serotyping.Of the 67 isolates identified from theaboriginal communities, 65 (97%)were from skin sites. Twenty-eight (42%)isolateswererepresented byM types Ml, M4, M52,

M53/80, M55, M57, M61/PT3875, M63, M80, and NZ1437. Theremaining39(58%)wereMNT.M-typingresults for the isolatesaregiveninTable 1.Isolates fromacute glomerulo-nephritis cases were identified as M53 (one case), M53/80 (one case),M55 (one case),and M57(two cases).

Of the15streptococcifromhospitalized patients,Mtypes M6, M12, M53, 61/PT3875, and M80 were represented

amongtheseventypeableisolates. Eight (53%)wereMNT. PCRand DNAsequencing.DNAs from the referencetype strains for MtypesMl,M5, M12, M24, M49,andM57were

tested by PCR. In every case the 5' sequences of the

amplified products were found to match published DNA sequences(see references 10, 18, 23, 19, 9, and 14,

respec-tively)

(data

notshown). Inaddition,the5'DNA sequences of the Northern Territoryisolates typed as

Ml,

M4, M12,

and M57 were found to be 95 to 99% homologous to the

published

DNA sequences

(5,

10, 14,

23).

These results stronglysuggestthat the PCRprotocol usedin the present

studyamplifiedthe genesforMproteinwithhighspecificity.

Ananalysisof DNA sequences revealed that, asexpected,

MNT isolates areheterogeneous. Overall, 18 MNT isolates

werealsoinvestigatedbyusingDNAsequencing, andeight distinct STs were differentiated

(Fig.

1). The DNA

se-quences forseven MNT isolates obtainedfromcommunity

A in 1987 were identical and classified as STDONALD

(Table 1). ST548 was based on the sequence of isolate 89/548, and ST550 was based on the sequence of isolate

89/550. FourMNTisolates fromcommunityC in 1990were

designated ST90/85. Two MNTs were designated ST88/25. Except for ST88/25, each of the distinct STswasobtained from differentaboriginal communities.TheST88/25isolates

were obtained 2 years apart, suggesting that atleast some

STs remain stable. The two members ofST88/25 differed

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STDONALD

D N G K A I Y E R A R E R A L Q E L G P V P R S L W L

GATAATGGCAAAGCTATATACGAAAGAGCTAGAGAAAGAGCCTTGCAAGAACTTGGACCTGTACCTCGTAGCTTATGGCTT

.-...

Q E

L

IT

K

K

L T E F E E K

Li

L

Q

N

D

Q

L L

82 CGAGAGTATGACAAAAATCAGGAGTTAACTAAAAAATTAACTGAATTTGAAGAAAAATTACTTCAAAATGATCAACTGCTA

S E N

|T

N K L N E L K A E K A Q V

IE

E K

LI

K E A R L N

163 TCTGAAAACACTAATAAACTTAATGAATTAAAGGCAGAGAAAGCTCAAGTTGAAGAGAAGTTAAAGGAAGCTCGACTTAAT

ST88/25

1

GATAGATATACCGATGCTCACAATGCAGTAACACAAGGGCGCACTGTACCGCTTCGGAATTTGTTACTTGAAATGGACAAA

:.N.

S K L R S

E

N E E L

O

A G L

O

E

K E

R E L E

"D

K"

82

AATAGCAAACTTAGATCGGAAAATGAAGAGCTCCAGGCTGGTTTACAGGAAAAGGAACGAGAGTTAGAAGACCTTAAAGAT

A E L K R L N E E R H D H D K R E A E

163 GCTGAGTTGAAGCGACTTAATGAAGAGAGACATGATCATGACAAAAGAGAAGCAGAG

ST548

1

ACTGAAGTTAAGGCTGCGGGGCAAAGCGCTCCTAAAGGTACAAACGTGAGCGCAGACCTATATAATTCGCTATGGGATGAA

82

AATAAAACTCTTAGAGAAAAACAAGAAGAGTATATAACAAAAATTCAAAATGAAGaGACAAAAAATAAAGAGTTAGATAAG

163

AAAAATAAAGAATTAGACTCTCGAGTTACAGATCTCATAGACGTAATTGAACATGACGAT

ST550

E

AP

K S

H

S I

N N E Q L IN E L N D LI

IE

E N N

b

1

GAGGCCCCAAAATCTCATAGTATTTCTAATAACGAGCAATTAATAAATGAATTAAATGACTTAATCGAGGAAAACAATGAC

82

CTTAAAGATAAACTTGCACGAAATCTAGATTTACTTGATAATACTAGAGAAAAAGATCCTCAGTACAGAGCATTAATGOGGG

163

GAAAATCAAGATCTC

ST9O/85

1

GAIGGTGPIGCGCGTTCCTAAGAATAATAGGCTATCGMAAAAATATAGTGAGCTATCGGAAAAATATGGTGCGCTATCGGPA

|KY IG

A LL D K Q

IG

A LL

D0K*S;

*

D

82

AAATATGGTGCGCTATTGGTAAAArGGCGCTATTGGATAAACAAGAAGAATTAGAAAAGGAAAATGAAAAGTTAGAT

163

TCTCAGGTTGCTGGTCTAATAGGTGTAGTTGAGAGTGATGAAGAAGAAGCTAAGCGCTCAAAGAACATGTACGAAACTTTC

STNS1

1

AGAGTTACTACTCGATCTCAGGCACAGGATGCAGCAGGATTAAAAGAAAAAGCTGACAAGTATGAGGTACGGAATCATGAG

t: *B

H

K T

E N S

D L K

L R I

V S

...

82

TTAGAACATAATAATGAGAAGTTAAAAACTGAGAATAGTGATTTAAAACTGAGA~ATAGTAAGT

ST1293

1

GCGGACCGTAAACTCGAAAAGCGTTTCTAATAGTAACGTGAGCATAAATCTATATAATGAGCTACAGGCTGAACATGATAAG

82

CTACAGACTAAACATAGGAGACTATATGGCTGTTAAAATAAAGACTAAA

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STNS14X

D N P S S V P V K K A A E L Y D K I K E L E

E

G R l

l-1

GACAATCCGAGCTCTGTCCCTGTCAAAAAGGCAGCAGAATTATATGACAAAATTMAAGMACTTGAAGAGGGAAGAGAAGAA

t t N D

|L

D K V K

E

E H K

K( v tff ,i

V K E E H K K DH

82

TTATTAAATGATTTAGATAAAGTTAAAGAAGAGCaTAAAAAAGATTTAGATAAAGTTWAGAAGAGCATAAWAAGATCAT

163 GAAAAACTTGAAAAA

FIG. 1. Nucleotide sequences of the eight distinct STs from the Northern Territory of Australia. The deduced amino acid sequences are indicated with single amino acid codes above thenucleotide sequence. The boxed regions indicate internal repeats, and the shaded areas indicate sequences that occur in another nontypeableM protein belonging to an ST as defined in this report. The sequence that is repeated in isolate 90/86 is underlined in theST88/25sequence.

slightly,in thatisolate90/86 had anadditional block ofrepeat

compared with the sequence of isolate of 88/25 (Fig. 1). Changesin the number ofrepeat blocks is a common feature

inMproteins.

The DNA sequences from three ofeight MNTs isolated

from hospitalized subjects did not match thesequences of the STs described aboveorpublishedsequences.TheDNA sequence for an isolate (NS1) which was associatedwith

toxic shock-like syndrome was the basis for another ST, STNS1. Likewise, ST1293 was assigned as the type for

isolate 1293, and STNS14X was assigned as the type for isolate NS14.

The derived amino acid sequences of the STs exhibit a

preponderance of glutamic acid residues, as isseen in the

published M-protein sequences. Also, in some STs

(STDONALD, ST548, ST550, ST90/85, ST1293, and

STNS14X),

blocks ofrepeats

containing

identicalor homol-ogousresidueswerefound

(Fig. 1).

Such blocks ofrepeats are common among M

proteins.

Although it is clear that all

thedesignated STswere differentfrom each other andfrom thepublished sequences of the genes forM

proteins,

there appearedtobe somesequencerelatedness between various STs, as follows: STDONALD and ST88/25 contain the

sequence ExDKN; ST88/25 and ST550 have a common

tetrapeptide

(DLKD),

ST548 and ST1293 contain the se-quenceNVSxxLYNxL, ST548 and STNS14Xbothhavethe sequenceKxLDK, ST90/85 and STNS1 share thecommon

sequenceELExxNEKL, ST90/85 andST1293 both contain the sequenceKxEEL, andST1293 andSTNS14Xshare the

tetrapeptide EELL.

DISCUSSION

The large number of MNT isolates obtained probably reflects the factthat the majority of GAS examined in our

studywereisolated from skin sites. Thesuccessof M

typing

is often less than30%insimilarsubtropicallocations of the world where skininfections areprevalent(16).

Further analysis of the M types of isolates was achieved

by amplifyingspecific DNAanddetermining thesequence.

The

specificity

of PCR was validated by determining the

DNAsequencesofamplified material from referencestrains. In addition, the 5' DNA sequences of some isolates that were M typeable matched the corresponding published se-quences.Thiswasconsistent with thefindingthat

serotype-specific epitopes are present at the N terminus of the M

protein (1, 2, 4). Basedifferences leading tochanges in the amino acid sequences were observed; however, these

changes presumably did not involve serotype-specific

epitopes.

The PCR method also

proved

useful in

defining

those

isolates that didnot reactwith referenceantisera. The DNA sequences of PCR products from MNT isolates did not

match publishedsequences, and STswereproposed onthe basis of the distinct sequence differences correspondingto

the5' sequence. We believe that the definedSTsobtained in the present study represent distinct M types for which

antiserawerenot available withinourlaboratories.

Among the isolates studied, we documented 20 distinct M typesbyacombination of methodsusing traditional

serotyp-ing andDNAsequenceanalysis.The M types present in one

community mostly differed from those present in another community, butthisprobably reflects the relative isolation that occurs betweenaboriginal communitiesinthe Northern

Territory. Nonetheless, thesameST(ST88/25)wasisolated from two distant communities 2 years apart. This may suggest that someSTs remain stable overtime,althoughthe number ofgenerations isnot known.

We conclude that DNAsequencing of PCR products is useful fordifferentiating amongisolates that possess the M

protein and that cannotbe typedby traditional serotyping methods. Characterization ofmore STs from agiven

geo-graphical areawillyield information abouttheevolutionary relationships of STs with each other and with the prevailing

Mtypes present in the samecommunity.Thesestudiesarein progress.

ACKNOWLEDGMENTS

We are grateful to David Ashbridge, Bart Currie, Paul van Buynder, Dave Kemp, and John Mathews for supplying the strep-tococcalisolates andcontributing inhelpful discussions. We thank Julie

Gaggin,

FrankHolland,and FranMorey for the identification of streptococci and Sue Walker for the serotyping. Elizabeth

O'Reilly provided excellent technical assistance. We thank the aboriginalpeopleand the healthcenterstaff from the communities where the studieswereundertaken.

These studieswere supportedby research grants from the Na-tional Health and Medical ResearchCouncilofAustralia.

REFERENCES

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136:2287-2292.

2. Beachey, E. H., J.M. Seyer,andJ.B. Dale. 1987. Protective immunogenicity and T lymphocyte specificity ofa tri-valent hybridpeptide containing the NH2-terminal sequencesoftypes 5, 6, and 24 Mproteins synthesized in tandem. J.Exp. Med. 166:647-656.

3. Brennan,R.E.,and M. Patel.1990.Acuterheumatic feverand rheumatic heart diseaseinarural AustralianAboriginal com-munity. Med. J. Aust.153:335-339.

4. Dale, J. B.,and E. H.Beachey.1986.Localization of protective epitopes of the amino terminus of type 5 streptococcal M

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5. Frithz, E., L.-O. Heden, and G. Lindahi. 1989. Extensive sequence homology between IgA receptor and M proteins in Streptococcus pyogenes. Mol. Microbiol. 3:1111-1119. 6. Gogna, N.K.,V.Nossar, andA.C.Walker.1983.Epidemic of

acute poststreptococcal nephritis in Aboriginal Communities. Med. J. Aust.1:64-66.

7. Gooder, H. 1961. Association ofa serumopacityreaction with serological typeinStreptococcuspyogenes.J. Gen.Microbiol. 25:347-352.

8. Griffith, F. 1934. The serological classification ofStreptococcus pyogenes. J. Hyg. 34:542-584.

9. Haanes, E., and P. P.Cleary.1989.Identification of a divergent Mproteingene and anMprotein-related gene familyin Strep-tococcuspyogenes serotype49.J. Bacteriol. 171:6397-6408. 10. Haanes-Fritz, E., W. Kraus, V. Burdett, J. B. Dale, E. H.

Beachey, and P. P. Cleary. 1988. Comparison of the leader sequences of four group A streptococcal M protein genes. NucleicAcids Res. 16:4667-4677.

11. Heath, D. G., and P. Cleary.1989. Fc-receptor and M protein genes ofgroupAstreptococciareproducts of gene duplication. Proc.Natl.Acad. Sci. USA 86:4741-4745.

12. Hollingshead, S. K., V. A. Fischetti, and J. R. Scott. 1986. Complete nucleotide sequence of type 6Mprotein of the group AStreptococcus. J. Biol. Chem. 261:298-301.

13. Jung, V., S. B. Pestka, and S. Pestka. 1990. Efficient cloning of PCR generatedDNAcontaining terminal restriction endonucle-aserecognition sites. Nucleic AcidsRes. 18:6156.

14. Manjula,B.N., K. M. Khandke, T. Fairwell,W.A.Reif, and K. S. Sriprakash. 1991.Heptad motifs within the distal subdo-main of thecoiled-coil rodregion ofMprotein from rheumatic fever andnephritis associated serotypes of groupAstreptococci are distinct from each other: nucleotide sequence of the M57 geneand relation of the deduced amino acid sequencetoother Mproteins. J.ProteinChem. 10:369-384.

15. Martin, D. R.,G. A.Meekin, and J.Finch. 1983. Streptococci-arethey differentinNewZealand? N.Z. Med. J. 96:298-301. 16. Maxted, W. R. 1980. Disease association and geographical

distribution of the M types of group A streptococcus, p. 763-777. In M. T.Parker(ed.), Streptococcal diseases and the

immune response. AcademicPress, Inc., New York.

17. Maxted,W.R., J.P.Widdowson, C. A.M.Fraser,L.C.Ball, andD. C.J.Bassett. 1973. Theuseoftheserumopacityreaction in the typing of group A streptococci. J. Med. Microbiol. 68:83-90.

18. Miller, L.,L.Gray,E.Beachey,andM.Kehoe. 1988.Antigenic

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19. Mouw,A. R.,E. H.Beachey,and V.Burdett. 1988. Molecular evolution ofstreptococcal M protein: cloning and nucleotide sequence of type 24Mproteingene and relationtoother genesof Streptococcus pyogenes. J. Bacteriol. 170:676-684.

20. Podbielski,A., B. Melzer, andR.Lutticken. 1991.Application of thepolymerase chain reactiontostudy the M protein(-like) gene

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23. Robbins, J., J. Spanier, S. Jones, W. Simpson,and P. Cleary. 1987.Streptococcus pyogenes type12Mprotein generegulation by upstream sequences. J. Bacteriol. 169:5633-5640.

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25. Saravani, G. A., and D. R. Martin. 1990. Opacity factor from group A streptococci is an apoproteinase. FEMS Microbiol. Lett.68:35-40.

26. Van Buynder, P., J. A. Gaggin, D. Martin, D. Pugsley, and J. D. Mathews. 1992.Streptococcal infection and renal disease mark-ers inAustralian Aboriginal children. Med. J. Aust. 156:537-540.

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0095-1137/93/071955-05$02.00/0

Letters

to

the Editor

Identification of

M

Types

of

Group

A

Streptococci

Itwaswithgreatinterest thatweread therecentarticleby Relfetal. (3) describingtheirapplicationof DNAsequence

analysistothe identification ofgroupAstreptococci (Strep-tococcus pyogenes). In their study, clinical isolates that were not M protein typeable bythe standard Ouchterlony double-diffusionmethod with antiseraagainstalimitedpanel of M types were investigated. They amplified M protein

genes (emm genes)of these strainsbythepolymerasechain

reaction and clonedtheproductsintoaplasmidvector, and

the 5' sections of the streptococcal DNA inserts were

sequenced. By this approach, 18 serologically

M-nontype-able isolateswereassignedtoeightdistinctsequencetypes. We have performed similar experiments and obtained

nucleotide sequence data from a number ofemm genes of strainsbelongingtovarious serotypes, includingthose ofM types8and 11. All of these serologicallywell-defined refer-ence strains and clinical isolates were obtained from the collection of the World HealthOrganization (WHO)

Collab-orating Centerfor Reference and ResearchonStreptococci,

Prague, Czech Republic. The sequence types designated ST550 and ST548 in the article by Relf and colleagues (3)

exhibit almost 100% homology to the sequenceswe deter-mined from the N-terminalregionsof theemm8 andemm11

genes, respectively.ThepublishedN-terminalsequences of ST550 and ST548 bothstart with the codon codingfor the mature Mprotein. Whencomparedwith thefirst 165

nucle-otides of ST550, our sequence differs only in the fourth nucleotideposition,while the first 120nucleotidesofST548

and our emm 11 sequence match completely. These

seg-mentsofemmgenes are knowntoencodethe type-specific epitopesof Mproteins,andrecently,wehaveshown that the

nucleotidesequencecorrespondingtotheN-terminal amino acids of themature Mproteinistypespecifically conserved

among epidemiologically unrelated strains, making this

re-gionanexcellent candidate foratype-specific hybridization

assay (2). Therefore,weconstructedoligonucleotide probes

of 30 'nucleotides in length derived from the emm 8 (or

ST550) and the emm 11 (or ST548) sequences that were

hybridized to dot blotted genomic DNA from group A

streptococcal strains of numerous serotypes as described previously (2). Therespective probes specifically identified

the five M 8 strains and seven M 11 strains being

investi-gated.

Therefore, we strongly believe that the sequences of ST548 and ST550 actuallyrepresent the N-terminal regions

of theemm11 andemm8genesand that theyshouldnotbe

describedassequencesfromM-nontypeable groupA

strep-tococci. Unfortunately, Relf et al. (3) did not test their isolates with M 8- and M 11-specific antisera. We would certainly appreciate the chancetoserotypethese isolates in theWHOlaboratoryinPrague.

Since many clinical isolates of group A streptococci,

especially those isolated from skin infections or in certain

areasoftheworld, e.g.,inAsia (1),cannotbe serotyped by

conventional means,the tools of molecular biology,

includ-ingthe approach ofsequencing signature portions ofemm

genes, willimprove epidemiologicalinvestigations ofgroup Astreptococcal infections. However,wewant toemphasize

that care should be taken not to designate a strain M

nontypeable by serologyunlessagivenisolate is testedwith the entirearrayof establishedM-typingsera.

REFERENCES

1. Kaplan, E. L., D. R. Johnson,P.Nanthapisud,S.Sirilertpanrana, and S. Chumdermpadetsuk. 1992. A comparison of group A

streptococcalserotypesisolated from theupperrespiratorytract

in the USA and Thailand: implications. Bull. W.H.O.

70:433-437.

2. Kaufhold, A.,A.Podbielski,D. R.Johnson,E. L.Kaplan,andR.

Lfltticken. 1992. M protein gene typing ofStreptococcus

pyo-genes by nonradioactively labeled oligonucleotide probes. J.

Clin. Microbiol. 30:2391-2397.

3. Relf, W. A., D.R.Martin, and K. S.Sriprakash. 1992.

Identifi-cation of sequence types among the M-nontypeable group A

streptococci. J. Clin. Microbiol. 30:3190-3194.

AchimKaufhold AndreasPodbielski

Institute of Medical Microbiology TechnicalUniversity (RWTH)Aachen Pauwelsstrasse30, D-5100Aachen

Germany

PaulaKriz-Kuzemenska

WorldHealth Organization CollaboratingCenter

for Reference and ResearchonStreptococci

NationalInstituteof PublicHealth

100 42Prague10, Srobarova 48

CzechRepublic Author's Reply

In response to the letter of Kaufhold, Podbielski, and

Kriz-Kuzemenska, wecommentasfollows.

Mnontypeabilityisusuallyunderstoodasindicatingeither thatan organismlacks Mproteinorthat itbelongsto anM typeoutside therangeforwhich it has been tested.

Provid-ing,as wasthecase,thatapaperstatesclearlytherangeof

antisera used, there can be no confusion. Few, if any, national streptococcal reference laboratories carry a full

complementofantiseratothemorethan80 known Mtypes.

Thus, therewill always be a group ofuncommon strepto-cocci thatmaynotbedefinitively identifiedin anycountry. Infact,thestudyofKaplanetal.(1) (referredtointhe above letter) usedalimitedarrayofantisera.

We have takencareinourarticle (2)toensurethat isolates

grouped byDNAsequencingwere referred to assequence types (STs) and not M types. Where homology of the ST

with that of a known M type was established, this was indicated. Furthermore, we stated, "We believe that the definedSTs obtained inthepresentstudyrepresentdistinct

M types for which antisera were not available within our laboratories." By this statement we do not exclude the possibilitythatanyoneof theSTscouldrepresentanMtype

notcoveredbyourantisera. Thisappearstohave been the

case, and we are grateful to Kaufhold, Podbielski, and Kriz-Kuzemenska forhaving identified the likelyMtype of

twoofourisolates.

studies (unpublished) showed that types 53 and 80 and some STs cross-reacted with the typing sera, and on sequencing we found that there is considerable sequence similarity within the N-terminal regions of M proteins from these types. Thus, isolates scored as M nontypeable or M53 or M80because of this ambiguity are now identified as one or the other M type or as STs upon sequencing of their corresponding M genes.

Finally, the definitions of both M nontypeable and se-quence typesshouldbeconsidered provisional, andas sera for new types become available or an identification with uncommon but already defined sera is made, the data base foremm gene sequences will vastly increase. Such knowl-edge will contribute to the continuous epidemiological sur-veillanceof group Astreptococci.

REFERENCES

1. Kaplan, E. L., D. R. Johnson, P. Nanthapisud, S. Sirilertpanrana, and S. ChumdermpadetsuL 1992. A comparison of group A

streptococcalserotypesisolatedfrom theupperrespiratorytract in the USA and Thailand: implications. Bull. W.H.O. 70:433-437.

2. Reif,W. A., D. R. Martin, and K. S.Sriprakash. 1992. Identifi-cation of sequence types among the M-nontypeable group A streptococci. J. Clin. Microbiol.30:3190-3194.

W. A.Reif

K.S. Sriprakash

MenziesSchool of Health Research P.O.Boax 41096

Casuarina Australia

D. R.Martin

NewZealandCommunicable Disease Centre P.a Box50348

Porirua NewZealand

Abolish

Mycobacterium paratuberculosis Strain 18

Mycobacteriumparatuberculosis is aslowly growing (12 to16 weeks),mycobactin-dependent organism whichcauses

paratuberculosis (Johne's disease) in ruminants (5). There has beenaheightenedinterest in thisspecies because of the

suggestion that it may be associated with some cases of

Crohn'sdisease in humans(3). Discussions ofthebiology of this species invariably result in erroneous assignment of

characteristics to the species which aresupported by

pub-lished data. Careful review generallyreveals that the

erro-neous datawereobtained from strain 18.

Recent articles in the Journalof Clinical Microbiology by Coffinet al. (6)andKunzeetal. (9)continue this trend and

erroneously identify the infamous strain 18asM.

paratuber-culosis, when in fact the strain is Mycobacterium avium

serovar2.Thecontinueduseofthisorganismin M.

paratu-berculosis research erodesour knowledge and

understand-ing of this species.

During the 1920's, W. C. Hagan at Cornell University observed thatoneofhislaboratorystrains ofM. paratuber-culosis grew faster than other strains and had lost its

dependenceon mycobactin. Althoughthislaboratorystrain

had beenpassed from graduate student tograduate student

and these same students were also workingon M. avium,

therewerefewcriteria availableto definitively identifythis

species, and the possibilityofcross-contamination wasnot

consideredatthe time. Theavailability ofarapidly growing,

mycobactin-independentstrainofM.paratuberculosiswas a

major boontoresearch around the world.

InOctober 1939, this laboratory-adapted strainwas

pro-videdto theRegional Animal Disease Laboratory, Auburn, Ala., where it became known asU.S. Department of

Agri-culture (USDA) strain 18 (10).At the request of the Ameri-can Type Culture Collection (ATCC), itwas deposited by

the USDA as the "working type" and designated ATCC

12227. Because of its relative ease of cultivation, rapid

growth, and lack ofmycobactin dependency, strain 18 was

widely used in experimental studies, as well as in the production of antigens, mycobactin, and vaccines. ATCC requested several additionaldeposits, thelastbeing October 24, 1966(10).

Inadditiontorapid growthandmycobactin independence, strain 18had manyother differences from M. paratubercu-losis. The inability of this strain to cause disease in

rumi-nants resulted in fruitless efforts to associate mycobactin dependency and virulence. The differences between strain

18and M.

paratuberculosis

were sogreat that insidersbegan toquestionifthis strainwasreallyalaboratory-adapted field strainof M.

paratuberculosis

at all. However, the technol-ogytodetermine such wasnot

readily

available atthe time. Recognizingtheuncertainty regarding this strain, in1968 Merkal (10) officially withdrew strain 18 from ATCC as

unrepresentative of thespecies and replacedit with a bona

fide

wild-type

strainofM.

paratuberculosis

asthe"neotype strain," designated ATCC 19698. Despite this withdrawal, strain 18continuedto seewidespreaduse in

paratuberculo-sis research.

With the advent of DNAtechnology, suspicions regarding

theauthenticityof strain 18wereconfirmed.Every studyin

whichstrain18 was used unequivocally showed that strain

18 was not a strain ofM.

paratuberculosis

but rather was M. avium(4, 7, 12). AlthoughM. aviumandM.

paratuberculo-sisareverycloselyrelatedand oftendifficulttodistinguish,

the absence of the

species-specific

insertion sequence

(IS900)in strain18, which exists in 15 to 25copiesin all M.

paratuberculosisstrains(8),confirmed that it could not have evolved or been laboratoryadaptedfrom M.

paratuberculo-sis.Even thearticles ofCoffinetal. (6)and Kunzeetal. (9)

serve toconfirmthetrueidentityof strain 18. Aswehadlong suspected,strain 18 isactually M.aviumserovar2andlikely