Polymerase chain reaction for identification of human and porcine spirochaetes recovered from cases of intestinal spirochaetosis

ELSEVIER

FEMS Microbiology

Letters 125 (1995) 225-230

Polymerase chain reaction for identification of human and porcine spirochaetes recovered from cases of intestinal spirochaetosis N.Y. Park, C.Y. Chung ‘, A.J. McLaren, R.F. Atyeo, D.J. Hampson

*

School of Veterinary Studies, Murdoch Uniuersiry. Murdoch, W.A. 6150, Australia Received 20 September

1994; revised 9 November

1994; accepted

10 November

1994

Abstract A polymerase chain reaction (PCR) amplification of 16s rDNA was developed to identify spirochaetes recovered from cases of intestinal spirochaetosis in humans and pigs; these bacteria belong to a distinct genetic group of spirochaetes, with the proposed name ‘Anguillina coli’. The PCR incorporated a universal eubacterial 16s rDNA sequencing primer (1492r), and a 21-base forward primer designed to include a nucleotide sequence specific for ‘A. coli’. The PCR was used to correctly identify DNA extracted from 43 isolates of ‘A. coli’ from humans and pigs, whilst no product was produced from Escherichia coli, or from other intestinal spirochaetes, including 38 isolates of Serpulina spp., and one each of Treponema succinifaciens and Brachyspira aalborgi. The amplification provided a rapid and simple means of identifying DNA from isolates of ‘A. coli’, and could be used on boiled whole ‘A. coli’ cells, with a detection limit equivalent to 2.5 X 10” cells. The reaction was used to detect and identify these spirochaetes from selective agar plates inoculated with stool specimens from infected pigs. Keywords: Intestinal spirochaetes;

Polymerase

chain reaction; Detection;

1. Introduction Intestinal spirochaetosis occurs in both humans and pigs [l]. In humans the condition is associated with a heavy colonisation of the large intestine by anaerobic spirochaetes which attach end-on to the epithelium of the colon and rectum 121. This colonisation has been considered to cause a variety of gastrointestinal disturbances, but especially long-

* Corresponding author. Tel.: ( + 61-9) 360 2287; Fax: ( + 61-9) 310 4144; e-mail: [email protected] ’ Present address: College of Veterinary Medicine, Chonnam National University, Kwang-Ju 500-757, Republic of Korea. Federation of European Microbiological SSDf 0378-1097(94)00502-S

Societies.

16s rDNA sequence;

Spirochaetes

standing diarrhoea and rectal bleeding [3-51. In pigs the infection causes a sloppy mucoid diarrhoea, a reduction in weight gain, and the spirochaetes again attach end-on to the colonic epithelium [6,7]. In humans, colonisation rates exceeding 30% may be found amongst individuals living in developing countries [8], in Australian Aborigines [9], and in homosexual males and AIDS patients in western societies [lO,ll]. Colonisation is uncommon in other people in western societies [9,12]. Recently, using multilocus enzyme electrophoresis (MEE) and DNA-DNA hybridisation, spirochaetes isolated from cases of intestinal spirochaetosis in humans and pigs have been shown to be closely related [1,13]. They form a genetic

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Microbiology

with the suggested name ‘Anguillina coli’ and are distinct from porcine intestinal spirochaetes in the genus Serpulina, including S. hyodysenteriae [13], the aetiological agent of swine dysentery [14], and the non-pathogenic human intestinal spirochaete Brachyspira aalborgi [15]. In the current study we have developed a technique for the identification of these potentially pathogenic organisms, from both humans and pigs, by amplification of a portion of their 16s rDNA using the polymerase chain reaction (PCR). group

Letters 125 (1995) 225-230

2.2. Microbial

culture and DNA extraction

[Ml,

2.

Materials and methods

2.1. Spirochaetes

Eighty-three strains of anaerobic intestinal spirochaetes were analysed. These bacteria included 38 strains of Serpulina spp., one each of Brachyspira aalborgi and Treponema succinifaciens, and 43 strains of ‘A. coli’ from humans and pigs. A summary of the identity and origin of the strains is presented in Table 1.

Table 1 Identity and origin of spirochaetes

The spirochaetes were grown in the trypticase soy broth-based pre-reduced anaerobic autoclaved liquid medium of Kunkle et al. [16], supplemented with 2% foetal calf serum and 1% cholesterol. Spirochaetes in late log-phase were pelleted from broth, washed twice in phosphate-buffered saline (pH 7.2), and resuspended in 3 ml of 50 mM Tris . HCl (pH 8.0), 50 mM EDTA, 20% sucrose. DNA was prepared as previously described [17]. DNA from T, succinifaciens 6091, grown in RTY medium [18], was a gift from Dr. T.B. Stanton, National Animal Diseases Center, Ames, IA. DNA from Escherichia coli strain JM109 was donated by B.C. Combs, Murdoch University. DNA concentrations were measured spectrophotometrically [19]. 2.3. Preparation

of whole cells for PCR

Dilutions of cells of human ‘A. coli’ strain HRM2[5], which had been washed twice in phosphate-buffered saline, were made from 1 X lo6 to 1 X 10’ cells, each resuspended in 1 ml of sterile

tested, and results of PCR amplification

Identity

Origin

Number of strains

‘Serpulina jonesii’ ‘Anguillina coli’ ‘Anguillina coli’ ‘Anguillina coli’ ‘Anguillina coli’ ’Anguillina coli’ ‘Anguillina coli’ Brachyspira aalborgi Serpulina hyodysenteriae Serpulina hyodysenteriae Serpulina hyodysenteriae Serpulina hyodysenteriae Serpulina hyodysenteriae ‘Serpulina intermedius’ ‘Serpulina intermedius Serpulina innocens Serpulina innocens Serpulina innocens ‘Serpulina murdochii’ (Group B spirochaetes) Treponema succinifaciens

Human, California Humans, Italy Humans, Australia Pigs, UK Pigs, Canada Pigs, USA Pigs, Australia Human, Denmark Pigs, UK Pigs, USA Pigs, Canada Pig, the Netherlands Pigs, Australia Pig, UK Pigs, Australia Pig, UK Pig, USA Pigs, Australia Pigs, Australia Pig, USA

1 5 17 2 2 1 15 1 2 5 3 1 10 1 4 1 1 4 6 1

+ , DNA amplified from all strains; - , no DNA amplified from any strains.

PCR results

_ -

PCR results

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distilled water. The tubes were placed in a boiling water bath for 10 min, centrifuged at 15 000 rpm for 10 min in a microfuge and the supernatants were discarded. Cell pellets were resuspended in 200 ,ul of TE buffer (10 mM Tris, 1 mM EDTA, pH 7.5). 5 ~1 from each tube was subjected to PCR. 2.4. PCR amplification

of 16s rDNA

The primers used in the PCR comprised a 22-base universal eubacterial 16s rDNA sequencing primer (‘1492r’) [20] with the sequence 5’-TACGGCTACCTTGTTACGACTT-J, and a 21-base forward primer (‘Acl’) sequence extending from a base position equivalent to position 200 on E. cofi 16s rDNA. This primer has a nine base difference from the type strain of S. hyodysenteriae (A.J. McLaren and D.J. Hampson, unpublished observations), and has the sequence 5’-AGAGGAAAG l-JTJTTCGC-l-K-3’. Template DNA (100 ng, except for boiled whole cells) in a total volume of 25 ~1 was amplified by PCR using 50 ng of each primer, in a 10% reaction buffer containing 2.5 nmol of each dNTP (Pharmacia), 1.5 mM MgCl, and 0.55 units of thermostable DNA polymerase (Tth plus, Biotech International, Australia). The thermal cycling consisted of an initial denaturation period (3 min, 94”(Z), then 30 cycles of annealing (45 s, 48”C), elongation (2 min, 72°C) and denaturation (1 min, 94”(Z), with a final 10 min extension period at 72°C. The PCR products were subjected to electrophoresis in 1.5% agarose gels in Tris-borate buffer (0.089 M Tris, 0.089 M boric acid, 0.002 M EDTA, pH 8.0) at 80 V for 50 min. The gels were stained with ethidium bromide and photographed under UV light. 2.5. Detection

of spirochaetes

mary plates was resuspended in 1 ml phosphatebuffered saline, and the DNA was extracted by a guanidine thiocyanate-diatomaceous earth protocol [21]. The cells were pelleted by centrifugation at 13 000 X g for 1 min, and 1 ml of lysis buffer (5 M GuSCN, 22 mM EDTA, 0.65% Triton X-100, 0.05 M Tris, pH 6.4) and 50 ~1 of 200 mg ml- ’ diatomaceous earth (DE Fluka) suspension in sterile distilled water was added. The solution was mixed thoroughly, allowed to stand for 10 mitt, then the DE with bound DNA was pelleted by centrifugation at 13 000 X g for 10 s. The pellet was washed twice in wash buffer (5.5 M GuSCn, 0.05 M Tris, pH 6.4), twice in cold (4°C) 70% ethanol and once in cold acetone. The pellet was dried at 55°C 100 ~1 of buffer (10 mM Tris, pH 8.0, 1 mM EDTA, 10 mM NaCl) was added, and the tube placed in a waterbath at 65°C for 15 min to elute bound DNA. The liquid was collected and subjected to the PCR as previously described.

3. Results A PCR product, approx. 1300 bp in length, was generated from the DNA of all the 43 human and porcine strains of ‘A. coli’, including the so-called ‘Serpulina jonesii’. No product was generated from

1

2

345

6

78

from porcine faeces

Faecal samples were obtained from 10 grower pigs in a piggery in a section of a shed where growth rates were depressed and mucoid faeces had been observed. The samples were cultured anaerobically at 37°C on 5% sheep blood agar plates containing and 25 /Lg ml- ’ each 400 pg ml-’ spectinomycin of colistin and vancomycin. Weakly haemolytic spirochaetes were identified on plates from two samples, and were subcultured and identified by MEE [6]. After 6 days, growth from each of the 10 pri-

Fig. 1. 16s rDNA from intestinal spirochaetes, amplified in PCR. Lane 1, size markers: Hind111 digested A phage DNA and HindIII/EcoRI-digested A phage DNA (Bresatec, South Australia). Lanes 2-5, product amplified from human (2, 3) and porcine (4, 5) strains of ‘Anguillina coli’: lane 2, HRM7; lane, WesB; lane 4, P43/6/78; lane 5, 7082; lane 6, Serpulina hyodysenteriae strain P18A; lane 7, Serpulina innocens strain B256; lane 8, Brachyspira aalborgi ATCC 43994. The bar shows the position of the 1375-bp marker, just above the amplified 16s rDNA in lanes 2-5.

228

any of the other spirochaetes

N.Y. Park et al. /FEMS

Microbiology Letters 125 (1995) 225-230

(Fig. l), or from E.

coli strain JM109. A PCR product of the correct predicted size was generated from 5 ~1 samples of each of the dilutions of HRM2 between lo6 and lo4 cells. This gave a theoretical detection limit equivalent and 2.5 X lo* boiled cells. PCR product of the correct size was generated from the bacterial growth on five of the 10 selective agar plates inoculated with porcine faecal samples. Spirochaetes were isolated from two of these samples, and confirmed to be ‘A. coli’ by MEE.

4. Discussion Amplification of the 16s rDNA gene by PCR has been used in the identification and detection of a variety of different bacterial pathogens [22]. The protocol developed in the current study was shown to be 100% sensitive and specific in that it correctly identified DNA only from all those intestinal spirochaetes belonging to the proposed group ‘A. coli’ [6], whether they were isolated from humans or pigs. This result confirms that these spirochaetes form a coherent genetic group, with a common 16s rDNA signature sequence corresponding to primer Acl. The fact that product was generated from the so-called ‘Serpulina jonesii’ confirms the results of multilocus enzyme electrophoresis, in which this spirochaete clustered with other isolates of ‘A. coli’ [l]. The ‘A. coli’ isolates were also distinct from the non-pathogenic human intestinal spirochaete Brachyspira aalborgi, from pathogenic and nonpathogenic porcine intestinal spirochaetes of the and from the non-pathogenic genus Serpulina, porcine intestinal spirochaete Treponema succinifaciens. This was important since these spirochaetes also colonise the large intestine, and have many phenotypic characteristics in common with ‘A. coli’

[1,6,131. The PCR was also capable of detecting DNA from boiled cells of ‘A. co/i’, thereby removing the necessity of prolonged DNA extraction. Using this technique, DNA from an equivalent of 250 cells was successfully amplified. The limits of detection in clinical samples were not established, but DNA was amplified from the primary bacterial growth on se-

lective agar inoculated with 5 of 10 porcine faecal samples, recovered from pigs in a herd with on-going problems of depressed growth rate and mucoid diarrhoea. Isolates of ‘A. coli’ were recovered from two of the plates, suggesting both that the PCR was correctly identifying ‘A. coli’ isolates, and that the procedure could detect lower numbers of these organisms than bacterial culture. Although this technique had the disadvantage that primary bacterial culture still was required, it avoided difficulties associated with the presence of polymerase inhibitors in faecal samples, and has the potential to be used as a sensitive and specific means of identifying human and porcine isolates of ‘A. coli’ in clinical specimens.

Acknowledgements This work was supported by grants from the Australian Research Council and from the Australian Pig Research and Development Corporation. N.Y.P. was in receipt of an International Reciprocal Fellowship with Korea, awarded by the Australian Research Council. Thanks are due to Dr. T.B. Stanton for the gift of T. succinifaciens DNA, and for helpful and stimulating discussions on intestinal spirochaetes. Dr. B.G. Combs kindly provided DNA from E. coli.

References [ll Lee, J.I. and Hampson, D.J. (19941 Genetic characterisation of intestinal spirochaetes and their association with disease. J. Med. Microbial. 40, 365-371. [21 Harland, W.A. and Lee, F.D. (1967) Intestinal spirochaetosis. Br. Med. J. 3, 718-719. a [31 Douglas, J.G. and Crucioli, V. (19811 Spirochaetosis: remedial cause of diarrhoea and rectal bleeding? Br. Med. J. 283, 1362. [41 Gad, A., Willen, R., Furug&d, K., Fors, B. and Hradsky, M. (1977) Intestinal spirochaetosis as a cause of long standing diarrhoea. Ups. J. Med. Sci. 82, 49-54. bl Sanna, A., Dettori, G., Agliano, A.M. et al. (1984) Studies of treponemes isolated from human gastrointestinal tract. Ig. Mod. 81, 959-973. 161Lee, J.I., Hampson, D.J., Lymbery, A.J. and Harders, S.J. (1993) The porcine intestinal spirochaetes: identification of new genetic groups. Vet. Microbial. 34, 273-285 [71 Taylor, D.J. (1992) Spirochaetal diarrhea. In: Diseases of

N.Y. Park et al. /FEMS

[8] [9]

[lo]

[Ill

[12]

1131

[14]

[15]

Microbiology Letters 125 (1995) 225-230

Swine, 7th edn. (Leman, A.D. et al, Eds.), Iowa State University Press, Ames, IA. 584-587. Barrett, S.P. (1990) Intestinal spirochaetes in a Gulf Arab population. Epidemiol. Infect. 104, 261-266. Lee, J.I. and Hampson, D.J. (1992) Intestinal spirochaetes colonising Aborigines from communities in the remote north of Western Australia. Epidemiol. Infect. 109, 133-141. Kasbohrer, A., Glederblom, H.R., Arasteh, K. et al. (1990) Intestinale spirochatose bei HIV Infektion. Vorkommen, Isolierung und Morphologic der Spirochaeten. Dtsch. Med. Wochenschr. 115, 1499-1506. Jones, M.J., Miller, J.N. and George, W.L. (1986) Microbiological and biochemical characterisation of spirochetes isolated from the feces of homosexual males. J. Clin. Microbial. 24, 1071-1074. Tompkins, D.S., Foulkes, S.J., Godwin, P.G.R. and West, A.P. (1986) Isolation and characterisation of intestinal spirochaetes. J. Clin. Pathol. 39, 535-541. Lee, J.I., McLaren, A.J., Lymbery, A.J. and Hampson, D.J. (1993) Human intestinal spirochetes are distinct from Serpulina hyodysenteriae. J. Clin. Microbial. 31, 16-21. Stanton, T.B. (1992) Proposal to change the genus designation Serpula to Serpulina gen.nov. containing the species Serpulina hyodysenteriae comb.nov. and Serpulina innocens comb.nov. Int. J. Syst. Bacterial. 42, 189-190. Hovind-Hougen, K., Birch-Andersen, A., Henrik-Nielsen, R. et al. (1982) Intestinal spirochetosis: morphological charac-

[16]

[17]

[18]

[19]

[20]

1211

I221

229

terisation and cultivation of the spirochete Brachyspira aalborgi gen. nov. sp. nov. J. Clin. Microbial. 16, 1127-1136. KunkIe, R.A., Harris, D.L. and Kinyon, J.M. (1986) Autoclaved liquid medium for propagation of Treponema hyodysenteriae. J. Clin. Microbial. 24, 669-671. Combs, B.G., Hampson, D.J., Mhoma, J.R.L. and Buddle, J.R. (1989) Typing of Treponema hyodysenteriae by restriction endonuclease analysis. Vet. Microbial. 19, 351-359. Cwyk, W.M. and Canale-Parola, E. (1979) Treponema succinifaciens sp. nov., an anaerobic spirochete from the swine intestine. Arch. Microbial. 122, 231-239. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. p. ES. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Lane, D.J. (1991) 16S/23S rRNA sequencing. In: Nucleic Acid Techniques in Bacterial Systematics (Stackebrandt, E., Goodfellow, M., Eds.), pp. 115-175. John Wiley and Sons, Chichester. Jones, G.F., Ward, G.E., Murtaugh, M.P., Lin, G. and Gebhart, C.J. (1993) Enhanced detection of intracellular organisms of swine proliferative enteritis, ileal symbiont intracelluloris, in feces by polymerase chain reaction. J. Clin. Microbiol. 30, 2611-2615. Persing, D.H., Smith, T.F., Tenover, F.C. and White, T.J. (1993) Diagnostic Molecular Microbiology Principles and Applications. pp. 173-308. American Society for Microbiology, Washington, DC.