Pulsed-field gel electrophoresis for sub-specific differentiation of Serpulina pilosicoli (formerly ‘ Anguillina coli’)

MICROBIOLOGY LETTERS

ELSEVIER

FEMS

Microbiology

Letters

141 (1996) 77-81

Pulsed-field gel electrophoresis for sub-specific differentiation Serpulina pilosicoli (formerly ‘Anguillina coli’)

of

R.F. Atyeo, S.L. Oxberry, D.J. Hampson * School of Veterinary Studies, Murdoch University, Murdoch, WA 6150, Australia Received 9 January

1996; revised version received 13 March

1996; accepted 25 March

1996

Pulsed-field gel electrophoresis (PFGE) was developed for subspecitic differentiation of Serpulina pilosicoli, and was applied to 52 isolates recovered from cases of intestinal spirochaetosis (IS) in pigs, dogs, human beings and various avian species. The technique was highly sensitive, differentiating the isolates into 40 groupings. Only six groups contained more than one isolate; in live of these groups isolates with the same banding pattern were either from pigs in the same herds (four groups), or from humans in the same community: the sixth group contained two identical Australian porcine isolates from unrelated herds in different states. Overall S. pilosicoli isolates were genetically diverse, but in some cases isolates cultured from the same or different animal species were closely related. This suggested the likelihood of cross-species transmission, including zoonotic spread. PFGE was a powerful tool for epidemiological studies of S. pilosicoli and also allowed examination of genetic relationships between isolates. Keywordr: Serpulim pilosicoli; Pulsed-field gel electrophoresis;

Sub-specific differentiation;

1. Introduction Serpulina pilosicoli,

a newly described species of [l], was previously known as ‘Anguillina coli’ [2,3]. These bacteria colonise the large intestine and cause a diarrhoeal disease called intestinal spirochaetosis (IS) in pigs [2,4], human beings [3], dogs [5] and birds [6]. Experimentally, isolates from pigs and human beings have been used to reproduce IS in chicks [7]. The bacteria can be identified on morphological and biochemical criteria [4,8], by a polymerase chain reaction [9], or by

intestinal

spirochaete

* Corresponding author. Tel.: +61 (9) 360 2287; Fax: +61 (9) 310 4144. E-mail: [email protected] 0378-1097 /96/%15.00 0 1996 Federation PIISO378-1097(96)00212-l

of European

spirochaetosis

using a monoclonal antibody against a specific 29 kDa outer membrane protein [lo]. A collection of 131 isolates of S. pilosicoli from human beings, pigs and a dog previously has been analysed by multilocus enzyme electrophoresis (MEE) [3]. The isolates were shown to be relatively diverse, being divided into 73 electrophoretic types (ETs). Although certain porcine and human isolates were closely related, none of the ETs included isolates from both these species. The single canine isolate was closely related to strains from human beings living in the same community. Restriction fragment polymorphism analysis and DNA-DNA reassociation assays on unclassified strains of intestinal spirochaetes from human beings and dogs also has shown

Microbiological

Intestinal

some

of them

Societies. All rights reserved

to be closely

related

[5,11].

78

R.F. AQYO et al. I FEMS Microbiology Letters 141 (1996) 7741

The aim of the current study was to develop pulsedfield gel electrophoresis (PFGE) as an additional technique for subspecific differentiation and genetic analysis of populations of S. pilosicoli.

2. Materials and methods

2.1. Bacterial strains Fifty-two strains of S. pilosicoli were obtained from the collection held at the Australian Reference Laboratory for Swine Dysentery, School of Veterinary Studies, Murdoch University, Western Australia (Table 1). These included 30 isolates from pigs, 12 from human beings, 4 from dogs, 4 from commercial chickens, and one each from a duck and a rhea. The isolates originated from Australia (n = 26), North America (n= 19) Europe (n = 6) and the Middle East (n = 1). The spirochaetes were weakly haemolytic, and had previously been identified as S. pilosicoli on the basis of their morphology (46 periplasmic flagellae at each cell end), their biochemical reactivity (hippurate positive, lack of B-glucosidase activity), and their genetic grouping using multilocus enzyme electrophoresis [ 141. 2.2. Culture Isolates were grown in 250 ml of pre-reduced anaerobic Trypticase Soy broth (TSB) containing 2% fetal calf serum, 0.5% newborn calf serum, 0.002% cholesterol and 0.1% resazurin [12]. 2.3. Phenollchloroform polymerase

extraction of DNA jbr

chain reaction (PCR)

Cultures were harvested by centrifugation at 25 000 X g for 20 min, resuspended in 200 ml of phosphate buffered saline (0.14 M NaCl, 2.5 mM KHsPOd, 7.0 mM KzHPOb, pH 7.2), and centrifuged at 25OOOxg for 20 min. The washed pellet was suspended in 3 ml of Tris-EDTA buffer (50 mM Tris-HCl, 50 mM EDTA, pH 8.0) and 20% sucrose, and stored at -20°C. DNA was extracted from thawed cells (500 pl volume) as previously described [13], with DNA concentrations quantified spectrophotometrically.

2.4. PCR The PCR was carried out as previously described [9], except that a hot start procedure was used with a Chill Out Wax phase separator (M.J. Research, Adelaide, Australia). Half of the reaction buffer, the primers, magnesium chloride and half of the milliQ water was placed in the bottom phase, a 20 ul volume of Chill Out Wax was carefully overlaid and allowed to solidify, and the remaining reaction buffer and milli-Q water was placed in the top phase with the target DNA and the Tth plus enzyme (Biotech International, Australia). 2.5. Pulsed-field gel electrophoresis

(PFGE)

2.5.1.

Preparation of’ DNA from cells Bacterial cells (10’“)were harvested from culture by centrifugation (10000 Xg, 4°C 20 min), washed twice in PBS, and suspended in 350 pl of PIV buffer (10 mM Tris, 1 M NaCl, pH 8) at 4°C. They were mixed with an equal volume of 2% low melting point agarose in PIV buffer and poured into a sterile, prechilled mould. The agarose was allowed to set at 4°C for 30 min, and then placed in 2 ml of lysis buffer (6 mM Tris, 1 M NaCl, 100 mM EDTA, 0.5% Brij-58, 0.2% sodium deoxycholate, 0.5% sodium lauroyl sarcosine, 1 mg ml-’ lysozyme, 20 pg ml-l RNAse, pH 8) at 37°C for 16 h. The plugs were hardened at 4°C for 20 min, and placed in 2 ml ESP solution (0.5 M EDTA, 10% N-lauroyl sarcosine, 100 pg ml-’ proteinase K, pH 8) in a waterbath at 55°C for 2 h. The process was repeated after hardening the plugs at 4°C. The plugs were left in fresh ESP solution at 55°C for 16 h, hardened at 4°C and washed in six changes of TE buffer (10 mM Tris, 0.1 mM EDTA, pH 7.6) consisting of two changes of 2 h duration at 37°C and four changes each of 1 h duration at 37°C. The plugs were hardened at 4°C between each wash. Plugs were routinely stored in 0.5 M EDTA.

2.5.2. DNA restriction digest and pulsed-field gel electrophoresis

A portion of each plug was placed in two changes of 0.23 mM phenylmethylsulfonylfluoride in TE buffer at 55°C for 30 min, hardened at 4°C between each change, and then placed in three changes of ice-cold TE buffer for 30 min. The plug portions

79

RF. Atyeo et al. I FEMS Microbiology L.eiters 141 (1996) 77-81

were placed in 80 ul of restriction buffer (2 ul restriction enzyme Mlul, 78 ul of 10% restriction buffer) for 24 h at 37°C before being loaded into a prechilled 0.5 xTBE 1% agarose gel. The plugs were sealed with fresh low melting point agarose and the gel left at 4°C for 24 h before being loaded onto a contour-clamped homogeneous electric field-DR 11 system (BioRad Laboratories, Richmond, USA). Table 1 Origin of 52 isolates of S. pilosicoli with PFGE patterns PFGE pattern

2 4 6 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 21 28 29 30 31 32 33 34 35 36 37 38 39 40

l-40

Isolate

Species

Origin”

HRM7 89-223A 95/0438 16242-94 24072-93A Meyers K9-12 D9201243a;b;c UNL-3 S76 13316 31B HRM14 89-2005A;B 4742 H21 167 Rosie 2299 Karlos;WesB ATCC 49776 Gap 37 H60-2 Dog 17 1648 UNL-5;B359 0419.5 P43/6/18 9803;4661.3

Human

Italy Canada WA USA USA USA USA USA USA Holland Oman Italy Canada Holland WA WA NT WA USA NSW WA WA NSW USA

Qul Wand F 3295 88-3769 B1555a 888 R4 89-1069 Wand l-7 88-392 1772 1588.5 Gap 401

Pig Pig Dog Dog Dog Pigs Pig Duck Chicken Human Human Pigs Chicken Human Human Human Humans Human Human Human Dog Pig Pigs Pig Pig Pigs Chicken Pig Pig Pig Pig Pig Rhea Pig Pigs Pig Chicken Pig Human

Qld Scotland Qld;Tas

Qld WA Vie Canada USA Vie USA Canada WA Canada Holland

Qld NSW

BWA, Western Australia; NT, Northern Territory; NSW, New South Wales; Qld, Queensland; Tas, Tasmania; Vie, Victoria.

Fig. 1. PFGE pilosicoli.

banding

patterns

obtained

from 20 isolates of S.

The gel was subjected to electrophoresis at 180 V for 18 h at 12”C, with pulse time ramped from 1 to 20 s. The gel was stained with fresh ethidium bromide (1.22 ug ml-‘), examined over a UV transilluminator, and photographed. Images were scanned and analysed using the GelManager programme (BioSystematica, Pod rovinou, Czech Republic), which created a dendrogram from a matrix of band matching coefficients by the unweighted pair group method of arithmetic averages (UPMGA) clustering fusion strategy.

3. Results and discussion All 52 isolates were confirmed as being S. pilosicoli using PCR. This technique amplifies a portion of the 16s ribosomal RNA gene [9], and the results demonstrate its usefulness for rapid identification of members of this new species. Digestion of DNA from S. pilosicoli isolates with Mlul reproducibly yielded 8-15 large fragments that were clearly resolved by PFGE (Fig. 1). Forty distinct banding patterns were obtained for the 52 isolates (Table 1). Pattern 7 contained three porcine isolates from the same herd in the USA; pattern 13 contained two porcine isolates from the same herd in Canada; pattern 18 contained two isolates from Aboriginal children living in the same commu-

R.F Atyeo rt al. I FEMS Microbiology

80

18

.. .. . .. ..0s..............~..A 38 39 40

0.2

0.4

0.6

0.8

1.0

Fig. 2. Dendrogram showing percentage similarity amongst 52 isolates of S. pilosicoli, divided into 40 PFGE banding patterns. Porcine isolates are shown in plain lines, human isolates in bold lines, canine isolates in barred lines and avian isolates in hatched

lines. nity in the North of Western Australia [14]; pattern 24 contained two porcine isolates from the same herd in the USA; pattern 27 contained two Australian porcine isolates, one from Queensland and one from Tasmania; pattern 36 contained seven isolates from a Western Australian herd. All other patterns were represented by single isolates. These results clearly demonstrate the stability of the PFGE patterns, and show that the same strain may infect multiple individuals on a farm, or in a human community. No direct evidence was obtained for cross-species transmission of strains, but this is likely to have been influenced by the diverse geographical origin of the isolates: experimentally, strains of S. pilosicoli have been shown to infect and cause disease in other species [7]. Further large-scale studies are required to look for natural

Letters 141 11996) 77-81

cross-species transmission in situations where there is greater opportunity for this to occur. The dendrogram produced by analysing the PFGE patterns is shown as Fig. 2. At a 50% level of similarity, the patterns could be divided into four groups: patterns 1-17; 18-22; 23-39; 40. Isolates in the first group came from human beings, pigs, dogs and birds, all originating from various countries (Table 1). Isolates in group two came from human beings in different countries, and from a dog living in a remote Australian Aboriginal community [14]_ This isolate previously has been shown by MEE to be closely related to isolates from humans in the same community [3]. Isolates in the third group came from pigs and birds from several countries. The single isolate in group 4 came from a homosexual male in Sydney, Australia. These results confirm that there is considerable genetic heterogeneity amongst S. pilosicoli isolates, although some grouping of strains according to the species of origin was observed. For example, 73% of the 30 porcine isolates from different countries were located in group 3, and three of the four canine isolates were closely related to each other in group 1. In contrast, isolates from avian species and from human beings tended to be more diverse, each with representative strains in three of the four genetic groups. In summary, PFGE is a powerful technique for strain identification of S. pilosicoli, and, like MEE, can be used to obtain an estimate of genetic relatedness amongst strains from different sources. The technique should prove extremely useful for epidemiological studies, and, for example, could be used to determine the occurrence of natural cross-species/ zoonotic transmission of strains of S. pilosicoli, and to investigate their association with IS in various species.

Acknowledgments This work was funded by the Australian Pig Research and Development Corporation. Roslyn Atyeo was in receipt of a Postgraduate Scholarship from the Corporation. Thanks are due to Mohammad Feizabadi and Darren Trott for technical advice and assistance.

R.F. Atyeo et al. IFEMS

Microbiology Letters 141 (1996) 77-81

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[8]

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old specific pathogen free chicks: an animal model of intestinal spirochetosis. Infect. Immun. 63, 3705-3710. Fellstrom, C. and Gunnarsson, A. (1995) Phenotypical characterisation of intestinal spirochaetes isolated from pigs. Res. Vet. Sci. 59, 14. Park, N.Y., Chung, C.Y., McLaren, A.J., Atyeo, R.F. and Hampson, D.J. (1995) Polymerase chain reaction for identification of human and porcine spirochaetes recovered from cases of intestinal spirochaetosis. FEMS Microbial. Lett. 125, 225-230. Lee, B.J. and Hampson, D.J. (1995) A monoclonal antibody reacting with the cell envelope of spirochaetes isolated from cases of intestinal spirochaetosis in pigs and humans. FEMS Microbial. Lett. 131, 179-184. Koopman, M.B.H., Kasbohrer, A., Beckmann, G., van der Zeijst, B.A.M. and Kusters, J.G. (1993) Genetic similarity of intestinal spirochetes from humans and various animal species. J. Clin. Microbial. 31, 711-716. Kunkle, R.A., Harris, D.L. and Kinyon, J.M. (1986) Autoclaved liquid medium for propagation of Treponema hyodysenteriae. J. Clin. Microbial. 24, 669-671. Turner, A.K., Atyeo, R.F., Sellwood, R. and Hampson, D.J. (1995) Distribution of the smpA gene from Serpulina hyodysenteriae among intestinal spirochaetes. Microbiology 141, 2041-2046. 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.