FEMS Microbiology Letters 35 (1986) 283-287 283 Published by Elsevier

FEM 02482

DNA base compositions and base sequence relatedness of atypical Campylobacterjejuni strains

from clinical material

(DNA-DNA hybridization; nitrate-negative campylobacters; GCLO-2)

R.J. Owen and C, Dawson

National Collection of Type Cultures, Central Public Health Laboratory, 61, Colindale Avenue, London, NW9 5HT, U.K.

Received 5 March 1986 Revision received 20 March 1986

Accepted 1 April 1986

1. SUMMARY

The relationships of nitrate-negative campylo- bacters (NNC) resembling Campylobacter jejuni were investigated by DNA base composition estimation (Tin method) and DNA-DNA hybridi- zation ($1 endonuclease assay). The 8 NNC strains which were from clinical material formed a homo- geneous DNA group with a high level of related- ness (approx. 70%) to typical (nitrate-positive) C. jejuni, but were less similar (approx. 35%) to Campylobacter coli, and only slightly related ( 10%) to Campylobacter fetus, Campylobacter laridis and Campylobacter sputorum. The NNC strains showed small but consistent genome differences from typical C. jejuni. As these differences can be correlated with several bacteriological test dif- ferences, we conclude that the NNC strains con- stitute a distinct subspecies within C. jejuni.

2. INTRODUCTION

Various Campylobacter-like organisms (CLO) that do not fully conform to the descriptions of recognised species within the genus Campylobacter

occur in human clinical material as well as in animals and the environment. Several reports in 1985 drew attention to the isolation of nitrate- negative strains resembling C. jejuni [1,2,3]. Six gastric Campylobacter strains, referred to as GCLO-2, were isolated in the F.R.G. from the antral epithelium of patients with upper gastroin- testinal tract symptoms [1], and apparently similar strains were isolated from the faeces of young children with gastroenteritis in central Australia [2,3].

These CLO were referred to as atypical C. jejuni [3] because of their inability to reduce nitrate, in possessing a single polar flagellum and poor or no growth at 42°C. However, in most other respects they clearly resembled C. jejuni and a high level of relatedness to that species was evident from DNA-DNA hybridization [2,3], cel- lular fatty acid analysis [4] and serology [2,3].

The purpose of the present study is to clarify, by means of DNA base composition estimation and DNA-DNA hybridization, the relationships between such nitrate-negative campylobacters (NNC) from different geographical locations, and to examine their DNA similarities to C. jejuni, C. coli and reference strains of other Campylobacter

0378-1097/86/$03.50 © 1986 Federation of European Microbiological Societies

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species. The results obtained show that the NNC strains are closely related to nitrate-positive C. jejuni, and constitute a distinct genomic group within that species.

3. MATERIALS A N D METHODS

3.1. Strains and growth conditions The 22 strains of Campylobacter used are listed

in Table 1. All strains were cultured on 5% (v/v) defibrinated horse blood agar under microaero- philic conditions in an anaerobic jar (catalyst re- moved) that was evacuated to a pressure of 56 cm of Hg and re-filled with a mixture of 10% CO 2 and 90% H 2.

Six gastric isolates of nitrate negative CLO were received from Dr. G. Kasper (Institute f'tir Hygiene und Med. Microbiologie, Klinikum der Statt, Mannheim, F.R.G.) and two representatives

Table 1

D N A base compositions and D N A - D N A hybridizations between nitrate-negative campylobacters and C. jejuni and other strains of the genus Campylobacter

Species and strain number a mol% G + C Relatedness b to D N A from

N N C strain C. jejuni NCTC11847 NCTC11392

Nitrate-negative campylobacters F.R.G. strains

NCTCl1847 27.6 100 68 NCTC11849 28.3 100 74 A197/84 28.7 100 68 A482/84 30.6 100 71 NCTCl1848 28.1 73 65

Australian strains A651/85 29.4 100 77 A652/85 28.3 88 61

U.K. strain A608/85 28.3 100 68

C. jejuni NCTCl1392 biotype I c 32.3 [5] 81 100 A198/81 biotype I ND d 78 95 A200/81 biotype I 30.7 [10] 75 81 [10] A202/81 biotype II 30.6 71 79 A195/81 biotype II 31.8 [10] 64 79 [10] NCTCl1351 biotype I 31.6 [5] 54 75

C. coli A176/81 32.3 [10] 45 39 A201/81 33.8 [10] 40 50 NCTCl1366 T 32.3 [5] 34 39 A193/81 31.2 [10] 33 19 NCTCl1353 32.9 [5] 28 38

C. laridis A24/81 31.2 7 21

C. fetus ssp. fetus c NCTC10842 T 36.3 [5] 10 16 [10]

C. sputorum ssp. bubulus NCTCl1367 32.3 [5] 8 29

" Strains were from NCTC (National Collection of Type Cultures, London). Numbers prefixed with 'A ' are field strains. The sources of origin of the N N C strains are given in the text and details on the other strains are given elsewhere [10]. 'T ' indicates type strain.

b All values are corrected for zero-time binding. Reproducibility on repetition of the same hybridization mixture was 7%. c Lior [11] biotype. d Not determined. e Type species of the genus.

of the gastroenteritis strains were received from Dr. T.W. Steel (Institute of Medical and Veterinary Science, Adelaide, South Australia). The details on isolation, source and conventional biochemical test characteristics of the strains are described fully elsewhere [1-3]. Also included here is a U.K. isolate of nitrate-negative CLO, which was re- ceived from Dr. Jane Symonds (Russells Hall Hospital, Dudley, U.K.).

3.2. DNA isolation Bacterial cells from 5 blood agar plates were

collected in 0.15 M NaC1, 0.1 M EDTA, (pH 8.0) containing 0.5 M sucrose, and the DNA was puri- fied and isolated as described previously [5]. The stock DNA solutions at a concentration of at least 50/~g/ml were stored at - 3 0 ° C .

3. 3. Estimation of DNA base composition The DNA base composition (mol% G + C) was

estimated from the thermal denaturation tempera- ture (Tm) as described previously [6,7], either in 0.33 x SSC (SSC = 0.15 M NaC1, 0.015 M tri- sodium citrate) or in 0.1 x SSC buffer. The base composition was expressed relative to a value of 51.8 mol% G + C for Escherichia coli NCTC9001.

3.4. DNA hybridization Purified DNA from NNC strain NCTC11847

and C. jejuni NCTCl1392 were labelled in vitro by the nick translation technique as described previously [5]. Hybridization mixtures (60.5 #1) containing 35 # g / m l of fragmented unlabelled DNA and 10 #1 of tritium-labelled probe DNA were denatured at 85°C for 10 min, then reassoci- ated for 20 h at 62°C (an intermediate tempera- ture stringency criterion) in Eppendorf tubes (1 ml) at a final salt concentration of 0.42 M Na ÷. Hybrids were assayed by the single-strand specific nuclease reaction [8] using about 3 units S1 endo- nuclease (Sigma) per hybridization mixture for 20 min at 50°C. The amount of radioactivity in double-stranded (Sl-resistant) form was deter- mined on Whatman DE81 discs [9]. Percentage hybridization was expressed relative to the ho- mologous reaction after correction for zero-time binding [7].

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4. RESULTS

4.1. DNA base compositions of NNC strains The DNA base compositions of the 8 NNC

strains were between 27.6 and 30.6 mol% G + C. The mean and standard deviation for the group was 28.7 + 0.9 mol% G + C (see Table 1).

4.2. DNA-DNA hybridization among NNC strains Table 1 shows that labelled DNA from NNC

reference strain NCTC11847 hybridized with DNA from 7 other NNC strains at relatedness levels of 73% and above. The mean hybridization for these strains relative to NCTCl1847 was 94 + 11% (Ta- ble 2).

4.3. DNA-DNA hybridization between NNC and C. jejuni

Labelled DNA from NNC reference strain NCTC11847 hybridized with 6 C jejuni strains (Lior biotypes I and II) to levels between 54-81% (Table 1) with a mean relatedness of 71 + 10% (Table 2). When labelled DNA from C jejuni NCTC11392 (Lior biotype I) was hybridized with the 8 NNC strains, DNA relatedness values were between 61-77%, with a mean value of 69 + 3%. The pooled result for hybridizations between both sets of DNAs was 70 + 7% (Table 2). Reciprocal hybridizations between the two labelled DNA probes (NCTC11847 x NCTC11392) gave a mean relatedness of 74 + 7%. Their difference was con- sistent with a mean reproducibility of 6% for the

Table 2

Summary of the tions within and bacters

percentage relative D N A - D N A hybridiza- between NNC and thermophilic campylo-

Nitrate-negative campylobacters (NNC)

C. jejuni

NNC 94+11 (7) a C.jejuni 70-1- 7 (14) b 82+ 8 (5) C coli 36+ 7 (5) 37+11 (5) C. laridis 7 (1) 21 (1)

a Figures in parenthesis are the number of cross-hybridizations between different pairs of DNAs.

b The reciprocal mean hybridization between the two probe DNAs (NCTC11847 and NCTC11392) was 74_+ 7%.

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techniques obtained in duplicate experiments in this study and previously [5,10,12].

4.4. D N A - D N A hybridization between NNC, C. jejuni and other campylobacters

The labelled D N A from N N C strain NCTC- 11847 hybridized with D N A from 5 C. coli strains at homology levels between 28 and 45% (mean 36 ___ 7%), but hybridized only at low levels with DNAs from reference strains of C. laridis, C. fetus ssp. fetus and C. sputorum ssp. bubulus (see Table 1). Reference D N A from NCTCl1392 C. jejuni was hybridized with the same set of test strain D N A and the results are given in Table 1. The levels of relatedness obtained were very similar to those of the N N C reference strain (NCTCl1847), and the average similarities between the main taxa are summarized in Table 2.

5. DISCUSSION

The D N A results summarized in Table 2 show that the nitrate-negative campylobacters (NNC) are a homogeneous and highly related group of strains (mean D N A homology 94%). No signifi- cant differences in base composit ions and D N A - D N A hybridization levels were observed between strains either from different geographical locations (F.R.G. Australia and U.K.) or from different types of clinical specimen (biopsy and faecal samples). However, small differences be-

tween strains were revealed in a study of restric- tion endonuclease digest patterns [12].

The overall D N A relatedness results confirm previous studies on the Australian strains [2,3] that N N C are closely related (approx. 70%) to C. jejuni, partly related (approx. 35%) to C. coli, but only slightly related (~< 10%) to selected repre- sentatives of other Campylobacter species. As the dot hybridization techniques used by Steele and colleagues [2,3] yielded relatively imprecise hy- bridization results, our aim was to obtain more accurate estimates of D N A relatedness at an inter- mediate temperature stringency criterion. We con- clude from the D N A data that the N N C strains should be classified in C. jejuni.

In agreement with Steele et al. [2], we believe the N N C strains warrant subspecies status within C. jejuni. The small but reproducible genomic differences revealed by the S1 endonuclease assay method between N N C and typical (i.e., nitrate- positive) members of C. jejuni (represented in this study by Lior biotypes I and II) were consistent with their previously reported phenotypic dif- ferences i.e. in nitrate reduction, growth at 42°C and flagella arrangement [1-3]. An additional dif- ference we have observed is that the N N C con- sistently have much weaker catalase activity than typical C. jejuni (A. Taylor, unpublished results). Therefore on phenotypic results, the N N C strains are clearly identifiable as a subspecies. Data on othe~ Campylobacter subspecies summarized in Table 3 show that the N N C and C. jejuni

Table 3

DNA relatedness within and between subspecies of Campylobacter

% Relatedness

Within subspecies Between subspecies

Range Mean Range Mean

C. fetus ssp. fetus a 75-100 90+ 5"~ 68-91 81+14 C. fetus ssp. venerealis a 91-97 93 + 3/

C. sputorum ssp. sputorum b 75-94 87 + 12 ~ 79-100 88 + 6 C. sputorum ssp. bubulus b 72-100 82+ 7/ C. jejuni (nitrate-positive strains) 64-100 c 82 + 8 C. jejuni (NNC) 73-100 94 + 11 1 54-81 70 + 7

a Calculated from data of Roop et al. [13]. Calculated.from data of Tanner et al. [14] and Roop et al. [15].

c These results agree closely with previously reported data on estimates of DNA heterogeneity within C. jejuni [13,16].

(n i t r a te -pos i t ive s t ra ins) are re la ted to each o the r at levels s imi la r to those of the 2 subspecies w i th in C. fetus ( the type species) a n d w i t h i n C. sputorum.

A C K N O W L E D G E M E N T S

W e wish to express our t h a n k s to Drs. J ane S y m o n d s , G . K a s p e r a n d T .W. Steele for k i n d l y

p r o v i d i n g s trains . Dr . B. Rowe is t h a n k e d for the b i o t y p i n g resul ts o n C. jejuni. Also we are grateful

to P. B o r m a n a n d A. Beck for t echnica l ass is tance.

R E F E R E N C E S

[1] Kasper, G. and Dickgiesser, N. (1985) Lancet i, 111-112. [2] Steele, T.W., Lanser, J.A. and Sangster, N. (1985) Lancet

i, 394. [3] Steele, T.W., Sangster, N. and Lanser, J.A. (1985) J. Clin.

Microbiol. 22, 71-74.

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[4] Goodwin, S., Blincow, E., Armstrong, J., McCulloch, R. and Collins, D. (1985) Lancet ii, 38-39.

[5] Owen, R.J. and Leaper, S. (1981) FEMS Microbiol. Lett. 12, 395-400.

[6] Owen, R.J., Legros, R.M. and Lapage, S.P. (1978) J. Gen. Microbiol. 104, 127-138.

[7] Owen, R.J. and Pitcher, D. (1985) in Chemical Methods in Bacterial Systematics (Goodfellow, M. and Minnikin, D.E., Eds.) pp. 67-93. Academic Press, London.

[8] Crosa, J.H., Brenner, D.J. and Falkow, S. (1973) J. Bacteriol. 115, 904-911.

[9] Popoff, M. and Coynault, C. (1980) Ann. Microbiol. Inst. Pasteur 131A, 151-155.

[10] Leaper, S. and Owen, R.J. (1982) FEMS Microbiol. Lett. 15, 203-208.

[11] Lior, H. (1984) J. Clin. Microbiol. 20, 636-640. [12] Owen, R.J., Beck, A. and Bornen, P. (1985) Eur. J. Epi-

dem. 1, 281-287. [13] Roop, R.M., II, Smibert, R.M., Johnson, J.L. and Krieg,

N.R. (1984) Can. J. Microbiol. 30, 938-951. [14] Tanner, A.C.R., Badger, S., Lai, C.-H., Listgarten, N.A.,

Visconti, R.A. and Socransky, S.S. (1981) Int. J. Syst. Bacteriol. 31,432-445.

[15] Roop, R.M., II, Smibert, R.M., Johnson, J.L. and Krieg, N.R. (1985) Can. J. Microbiol. 31, 823-831.

[16] Owen, R.J. (1983) Eur. J. Clin. Microbiol. 2, 367-377.