Detection of Leptospiraceae by amplification of 16S ribosomal DNA

FEMS Microbiology Letters ~)(1992) 267-274 ~3 1992 Federation of European Microbi,logical Societies 0378-1097/t~2/$O5.tH)

267

Publishedby Elsevier

FEMSLE {)4764

Detection of Leptospiraceae by amplification of 16S ribosomal DNA J.V. Hookcy PHLS Leptospira R,fer,'ncc l.aborau~r)', FAO / H'tlO Collaborating ('entre. ('purity thJ~pitaL th'r~for~L U.K.

Rccci~ed I1| Oclohcr Iq9 I Revision received 4 November I~lt~l Accepted 5 November It~lt~l

Key words: Leptospiraceac: Polymcrasc chain reaction: 16S rDNA

I. SUMMARY

2, INTRODUCTION

The polymerase chain reaction (PCR) was developed to detect Leptospiraccae. Primers wcrc used to amplify a 631 base-pair (bp) 5'-region of 16S rDNA. Representative strains from the species, Leptospira blterrogans senstt stricto, L. borgpeterseniL L. noguchii, L, santarosuL L. weilii, L. b~adai, L. meyeri and the single member strain of Leptonema were amplified. In contrast, strains representing the saprophytic species, L. bifiexa. L. wolbachii and L. part'a were not amplified. There was no PCR product from 23 phylogenctieally unrelated species of bacteria. As little as 10-1 pg of purified DNA and as few as I~1-1 leptospires could be detected using this PCR analysis, isolates of leptospires from ,:linical sources gave a positive PCR band, but those from surface waters did not.

T|lc family !.,:ptospiraccac comprise two gencra, Leptospira and Leptonema [t]. Leptonema is represented by the single species. Leptonema illini and the taxon Lepto~pira by the pathogens, L. interroguns .~ettstt stricto, L. borgpetersenii, L. noguchii, L. santaro.~ai, L. weilii and L. btadai and the saprophytic species, L. biflexa, L. meyeri, L. wolbachii and L. part'a [2]. The dctection of leptospires (Lepto.spira and L,,ptom'ma) in clinical and ew,,ironmental sampies remains limited and difficu!t. Conventional methods such as isolation by culture and subsequent dctection by dark-field microscopy are either too slow (up to 3 months) or unreliable [3]. Chromosomal DNA probes have been developed [41 yct they were neither specific nor sensitive (between 7511and 2501]organisms). An alternative approach is to use probes that are complementary to small ribosomal subunits (16S rRNAs). Oligonuclcotidc primers for use in the polymerasc chain rcaction (PCR) and complementary to defined regions of the 16S rRNA gene have been u~cd in the detection of mycobacteria [5].

('orreslnmdence to: J.V. ttookey. PILLS Leptospira Reference Laboratory, FAO/WHO Colklboraling Centre. Counly I-lospital, Hereford HRI 2ER, U.K.

208

The aim of this study was to develop a rapid, specific and sensitive method for detecting Lcptospiraceae based on the amplification of 16S rDNA.

3. MATERIALS AND METHODS

3.1. Bacterial strahts and culture procedmvs The strains used are given in the legends to Figs. 1 and 2. Strains of Leptosph'a and isolates from clinical and environmental origins wcrc ~ulturcd according to Ellinghausen and McCullough

[6]. 3.Z DNA preparatkm Chromosomal DNA was isolated and purified as previously described [7]. 3.3. Preparation of biotbz labelled 16S ribosomal DNA Plasmid DNA (plasmid pNOl311 contains a 734-bp 5'-3' Hindlll-Arali ~6S rRNA gcne fragment insert derived from pKK 3535 [8]) was extracted from cultured Escherichia colt NO2356 host cells according to the manufacturer's instructions (Cireleprep TM, Stratech Scientific, Bedfordshire L U 2 0 N P , U.K.) and 100 ng of

DNA labelled using a random-primers kit in the presence of biotin-I I-dUTP following the protocol given by the manufacturer's (Gibco BRL, Paisley, Scotland).

3.4. Amplification of 16S ribosomal DNA b), PCR PCR was performed on a Perkin-Elmer Thermal Cycler (Perkin-Elmer Limited, Buckinghamshire HP9 IQA, U.K.) in PCR tubes that contained approximately 1(]0 ng of DNA template and the reaction mix made up of 5 #1 of 10 × PCR reaction buffer (500 mM KCI, 100 mM Tris. HCI, pH 8.3, 35 mM MgCI2, I).1)1% gelatin), 100 #M each deoxynucleotide triphosphate (Pharmacia, Milton Kcynes MK5 8PH, U.K.), 511 pM each primer and the sequences for these were: 5'

C6C

T56

C6G

C6C

6T¢

TTA

AA 3'

(34... 53) and 3' AAG GTC CAt a r c 6cc ACT r 5' (647... 665 J, glycerol at 111%(v/v) (Gibco BRL}, 1.25 units of 'Amp[iTaq' DNA polymerasc (I.L.S. Limited, London ECIA 7HU, U.K.) and sterile "Millipure" water to 51)/zl. Following an initial 5-min incubation at 95°C, thermal cycling ( × 32 cycles) proceeded with each segment of one cycle being: 94°C for 40 s; 58"C

Fig. I. Agarose gel Ca) find Southern blot Ib; probed wilh biotin-labelled 16S ribosomal DNA} analysis of PCR-.'Lmplified product from leptoslfires. Lanes ! and 21 were biolin-QXI74 RF DNA.tlinfl fragment size-markers: lanes 2-14 were representative strains of LtTmJspira imerrogans sots, stri,'to: hme 2. AUSTRALIS tm.~tralis Ballico: 3. A bratishwa J~z-Bratislava; 4, ALISTRALIS .regis Fudge: 5. AUTUMNALIS autumnalis Akiyami A: 6, CANICOLA canicola Hond Utrecht IV: 7, (" schuc./f~wri Vleermuis ~)l)C; S. |IEBDOMADIS heh~hmmdis Hebdomadis: 9. H kremastos Kremastos: 10. I C T E R O | I A E M O R R I I A G I A E copcnhageni M 21); II. I icterohaemorrhagiae RGA (type strain, source U.K.): 12. I h'terohaemorrhagiae RGA (type strain, source Amsterdam): 13.

POMONA pomona Pomona: and 14, PYROGENES p.vrogene~ Salinem. Lanes 15-18 were representative strains of L. borglwwrsenii: 15. BALLUM balhon Mus 127; 16. JAVANICA jartoli('a Veldrat Batavia 46 (type slrain, source U.K.); 17 SEJROE sejrt~, M 84: and 18. TARASSOVI tarassoci Peripelicyn. Lanes 19 and 21)were str;.fins representing L. m~guchii: !0. LOUISIANA louisiana LSU 1945: and 2tl. PANAMA patmma CZ 214 (type strain, source U.K.). Lanes 22-25 were strains representing L. .~antarosai: 22, HEBDOMADIS bormcam~ HS 622: 23. SHERMANI sherma,i LT 281 (type strain, source Amsterdam): 24. TARASSOVI bakeri LT 79; and 25. TARASSOVI rama 316. Lanes 26 and 27 were strains represenling L. weilii: 26. CELLEDONI ce#edoni Ce#e,hmi (type strain, source U.K.); and 27, SARMIN swmin Sarmin. Lanes 28-31 were representative strains of L. hif/exa: 28 SEMARANGA patoc Paloc l (type: strain, source U.K.); 2% SEMARANGA patoe Patoc [ (source Italy); 311. ANDAMANA amlamana CH II (source Amsterdam); and 31. ANDAMANA amhtmana Ctt 1 (source France). Lanes 32 and 33 were representative strains of L. meyer/: 32. RANARUM rananon ICF (type str.'fin, source Amsterdam): and 33. SEMARANGA .wmamnga Veldral S.173. lane 34 L. wolbachii CODICE c&' Biflexu ~IDC (type s.'rain, source Italy): lane 35, L. inadai (type strain, source U.S.A.); lane 3fi, L. parca (lype strain, source Northern Irdand}; lane 37. la'plotlt.l~la i#ini (type strain. source Denmark): kme 38. ICTEROttAEMORRFIAGIAE icrerohaemorrhaghw lc|cro I (rico-type strain [17], source U.K.); hme 3g. I icwrohaemorrlutg, iae Iclero I (neo-type strain [17] source Amsterdam): ano lane -it). a negative control having no DNA template. 5/zl of amplified product were loaded to each well.

269

EDTA, pH 8.1) buffcrcd gel) at Ill0 mA for 2 h. Gels were stained, visualised and photographed as previously described [7]. Gels were blotted onto nylon membranes (1}.45 ,am pore size 'Hybond N'; Amersham International, Buckinghamshire HP7 9LL, U.K.) in the presence of 211x SSPE {saline-sodium phosphate-

for 17 s; and 72°C for 1 rain. An additional cycle at 72°C for 10 min was included as a final step. 3.5. Analysis of ampli]ied rDNA 5 or I0 `al of product were taken for analysis by gel electrophoresis (3.0c/r NuSieve GTG/ SeaKem GTG composite agarose Tris-acetatc1

926

2

3

4

5

6

7

8

9

10

tl

12

13

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15

16

17

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19

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713

~631

bp

~450

bp

553 500 427°

417,413 31t

926

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500 427,

.117,413 311

21

22

23

24

25

26

27

28

29

30

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32 33

34

35

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37 38 39 4 0

270

1

2

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22 23 24 25 26 27

28

10

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1314

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631 bp

=; atl~ ~

631

bp

21

29 30 31 32 33

Fi~,. I. (continued),

34

35 36

37 38 39 40

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271

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Fig. 2. Ag;u'os¢ gel analysis ~t P('R-amplified product fr(~m cJinical and environmental leptospire isolates and from phylogenetically ¢mrdated sources. Lanes I and 21 were OXI74 RF D N A - I l a ¢ ] l l fragment sizc-markcr.~; lane 2. L. hlterroqa..~ ~¢n.m .~trk'to ICTEROIIAEMORRIIAGIAE u'terohaemorrhagiae RGA (source Amsterdam and included in this assay as a ~'cak-positive ColllrOI); hines 3 and 4 were clinical is~dalcs: and lanes 5-9 were environmental Icpt~spire i.~tdales: hine Ill, llorrelia hur.~chprj'eri strain B31, ATCC *352111: lane i l. CmnpyhJbacter chtaedi N('TC ** + I 1~5~): I2. C. coil NCTC" 11351: 13. (: cryaerophiht NCT(" 11885; 14. C. ]'emwlliae NCTC I Ib13: 15, ('. h'tus ssp. ]'etu.~ NC'TC II)r,;42, If), C. jcitmi NC'TC 11351; 17, ('. jejuni NC'T(" 113t)2: 18. (: muco.~ali.~ NCTC I IO(Jl; I(,I, ih,licohacter pyl, ri NCTC 11638; 20, tl. pylori NCTC" 11911c 22, E,cherichht coli N(.'TC t,~(Hll; 23. llaemophilus #t]lm'nza (wild strain): 24. Kh'i,sh,lht pneum~mhw; 25, L,'gumella mic~ht~h'i strain TATLOCK; 2¢), L. pm'umophila NCTC I1192; 27, Listeria m~moc3't~Ltlem's (wild strain): 28, M.vcohacterium tubercuh~.~ix: 29, hL hori.~"(wild strain); 3(I. M. bori.~ I]CG: 31, Psetuhmuma., uenq,,ittosa; 32, Salttlottelht lyl~hb,urium; 33. Sttq~hylo~'occus aztreux; 34. Stn'ptocrn'cux pnelollottht NCTC 7L178:35. Yershfia emocolitica NCTC II 177; 36, |~bllim'lla recta N(.'TC 11489; 37. Sacx'harmuyce.~ ceretisuw; 38. herring sperm DNA; 39. human DNA; and ,.11),a negative control having no DNA template. 5/41 o[ itmplified prt~duct were loaded 1u e~|ch welt. * American Type Culture Coll,¢clion: * * National Collection of T~'pc Cultures.

272

I

2

3

4

5

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I0

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631 bp 450 bp

533, 427, 311

417,

413

631 bp

q26 bp 713 553. 427, 417,

413

.~II

11

12

13

14

15

16

17

18

19

20

Fig. 3. Agarose gel analysis of PCR-amplified product from L. hlterrogans st'nsu slricto I C T E R O H A E M O R R H A G I A E k'ten~haemorrhagiae RGA (source Amsterdam). Lanes 1 and 11 were OX174 RF DNA-IlaelI! and lanes l0 and 211 were biotin-f0XI74 RF DNA-Hinfl fragment size-markers, Lanes 2-8 were serial 10-fold dilutions of purified-DNA: and were ItS) ng (lane 2), 10 ng (lane 3), 1 ng (lane 4), 101) pg (lane 5), 10 pg (l:me 6), t pg (lane 7), 11111l~g (lane 8) and lane t) was a negalive control having no DNA-template. Lanes 12-18 were serial 10-fold dilutions of ~ suspension of I icterohaemorrhagiae RGA containing I x Iii 5 bacteria ml- t (lane 12), I × 1tl4 (lane 13), 1 × 103 (lane 14), ! X 111"~(lane 15), 1 X l0 t (l~ne 16). I x 10" (lane 171 ;md I × I11~i (lane 18) and lane 19 was a negative control having no DNA-template. 111t,tl of amplified product were loaded to each well.

273 EDTA, pH 7,4) according to standard methods [9]. Pre-hybridisation, hybridisation and post-hybridisation conditions followed that given by Owen, Costas and Dawson [10]. Hybrids were visualised using a colourimetric non-radioactive detection kit (BIuGENE TM) according to the manufacturer's instructions (Gibco BRL). 4. RESULTS AND DISCUSSION Leptospirosis and its aetiological zoonotic agent, L. interrogans senstt-htto [! I], have bccn the subject of rcccnt media interest with the apparent risk to those that come into contact with potentially contaminated waters, such as canoeists and other recreational water users. The development of a rapid, specific and sensitive method tor leptospire detection in clinical and environmental samples would bc of benefit in disease surveillance and epidcmiologicai invcstigations. PCR primers were chosen on the basis of published, albeit limited, 16S rRNA sequence [12] and catalog data [13]. As expected, a positive 631-bp PCR product of predicted size [12] was observed for strains representing the species L.interrogans sensu stricto (Fig. i. lanes 2-14), L. borgpetersenfi (Fig. I, lanes 15-18), L. noguchii (Fig. 1, lanes 19 and 2{)), L. santarosai (Fig. I. lanes 22-25), L, weifit (Fig. 1, lanes 26 and 27) and Lepto;~ema illini (Fig. 1, lanc 37) and a negative result was recorded for duplicatc strains tff L. bqlexa SEMARANGA patoc Paloe 1 (Fig. I, lanes 28 and 29L Since L, hiflt:ra is the nucleus for saprophytic strains [2] it was not unexpected to observe that a negative result was also obtained for the duplicate and closely related strain, L. biflexa ANDAMANA atuhmuma CH 11 (Fig. I, lanes 30 and 31). A faint and anomalous "ghost'-product of approximately 450 bp in size was visualiscd on agarosc-gels and Southern blots (Figs. 1, 2 and 3). This result indicates that mispriming events wcrc occurring within the target, yet it does not present a problem in this analysis since this band was not of the expected or even correct size. Other species of Lepto.~pira were also analysed. Negative results were obtained tbr the

saprophytic fi)rms, L. wolbadfii CODICE cdc Biflexa CDC (Fig. I, lane 34) and L. pan'a (Fig. l, lane 36). However, a positive 16S rDNA product was obtained from the single and pathogenic member of L. inadai (Fig. 1, lane 35) and from representative strains of L. meveri (Fig. 1, hines 32 and 33). The strains RANARUM ranamm ICF and ~.EMARANGA semamnga Veldrat S.173 were isolated from a leopard frog (Rana pipicus) in Iowa, U.S.A. [14] and from a rat (Rattu.~ blvt'ictuuhmt~ ) in Java [15], respectively, These str~ins are now regarded as being saprophytes on the sole basis of avirulcnce, however their physiological characteristics are that of both pathogenic and saprophytic forms [2,15], Their true affiliation and the significance of these PCR results is, therefore unclear. There was no amplification product from phylogenctically unrelated bacteria or from the yeast° Soccharom)'ces cerrcisiae, herring sperm DNA or even human DNA (Fig. 2), indicating that this PCR analysis may be [eptospire-specific. The sensitivity was between II) and i pg of purified DNA and between 10 and 1 lcptospires could be detected (Fig. 3). These limits of detection are a considerable improvement on that previously obtained using DNA probes [4]. Lcptospires isolated from clinical and environmental sources wcrc studied. The clinical isolates, identified as L. interrogans [16], gave a positive amplification product (Fig. 2, lanes 3 and 4), yet strains isolated from surface waters did not (Fig, 2, lanes 5-9). These environmental strains have provisiomdly been typed, using serologicaI methods, as being similar to L. biJh:ra (unpublished data). With the notable exceptions of strains representing the saprophytes, L. kiflexa, L. wolbachii and L. part'a, evidence has been provided that selected primers fi)r use in the PCR can lze used in the rapid (2 days from biomass to the analysis of a PCR product), specific and sensitive detection of Lcptospiraccae and lcptospirc isolates. ACKNOWLEDGEMENTS The author is indebted to colleagues who kindly provided cultures and chromosomal DNA

274 and to M a s a y a s u N o r m u r a , University o f California, lrvine, U.S.A. for supplying the v e c t o r carrying the plasmid pN01311.

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[8] Brosius, J., UIIrich, A., Raker, M.A., Gray, A., Dull, TJ., Gutell, R.R. and Noller, H.F. (1981) Plasmid 6, 112-118. [9] Sambrook, J,, Fritsch, E.F. and Maniatis, T. (1989) In: Molecular Cloning: A Laboratory Manual (Sambrook. J., Fritsch, E.F. and Maniatis, T., Ed.~.), pp. 9.31-9.62. Cold Spring Harbor Laboratories, Cold Spring Harbor, NY. [10] Owen, R.J., Costas, M. and Dawson, C. (1989) J. Clin. Microbiol. 27, 2338-2343. [1 I] Johnson, R.C. and Faine, S. (1984) In: Bergey's Manual of Systematic Bacteriology, Vol. I (Krieg, N.R. and Holt. J.G., Eds.), pp. 62-67, Williams and Wilkins, Baltimore, MD. [12] Fukunaga, M., Horie, l., Okuzako, N. and Mifuchi, !. (1990) Nucleic Acids Res. 18, 366. [13] Woese, C. (1987) Microbiol. Rev. 51, 221-271. [14] Diesch, S., McCulloch, W.F., Braun, J.L. and Ellingbausch, I',.C. Jr. (1966) Nature 209, 939-940. [15] Cinco, M. and Dougen, R. (1975) Int. J. Syst. Bacteriol. 25, 138-142. [16] Hookey, J.V. (1990) FEMS Microbiol. Left. 72, 329-336. [17] Kobayashi, Y., Sada, E. and Tamai, T, (1991) Microbiol. Immunol. 35, 67-76.