ELSEVIER

IMMUNOLOGY AND MEDICAL MICROBIOLOGY

FEMS Immunology and Medical Microbiology 14 (1996) 129-134

Comparison of random amplified polymorphic DNA analysis and enterobacterial repetitive intergenic consensus-PCR for epidemiological studies of Salmonella
Yves Millemann , Marie-Claude Lesage-Descauses, Jean-Pierre Lafont, Elisabeth Chaslus-Dancla *
Station de Pathologic Aviaire et Parasitologic, Institut National de la Recherche Agronomique, 37380 Monnaie, France 1996; accepted 22 February Centre de Tours-Nouzilly,

Received

16 January

1996; revised 22 February

1996

Abstract Discrimination of 70 Salmonellastrains previously studied by ribotyping was realized by RAPD and ERIC-PCR analysis. RAPD results on the 56 S typhimurium isolates did not closely match those of ribotyping. With ERIC-PCR, two fingerprints only were obtained. For the 14 S. enteritidis strains, a helpful discrimination was obtained with RAPD analysis, while ERIC-PCR resulted in a single fingerprint. Keywords:RAPD; ERIC-PCR; Ribotyping;
Epidemiology; Salmonella typhimurium; Salmonella enteritidis

1. Introduction Salmonella typhimurium and S. enteritidis are among the major etiologic agents of human gastroenteritis in France [l] and are also the serotypes most frequently isolated frolm poultry products. Epidemiological studies of Salmonella infections require the use of efficient molecular markers to trace precisely the diffusion of strains. Phenotypic and genotypic techniques have been used for subtyping Salmonella serotypes, including phage and bacteriocin typing, plasmid profile analysis, RFLP, ribotyping, IS200

* Corresponding author. Tel: +33 47 42 77 65; Fax: +33 47 42 77 74; E-mail: elisabeth.chaslus-dancla@tours.inra.fr Present address: Patholoaie du B&ail. DPASA. Ecole Nationale V&%inaire dAlfort, 94704 Maisons-Alfort Cedex, France. 09288244/96/$15.00 Copyright PZI SO928-8244(96)00021-l 0 1996 Federation of European

typing, pulsed-field gel electrophoresis, and recently arbitrarily primed polymerase chain reaction (APPCR) and ERIC-PCR [2-71. Random Amplified Polymorphic DNA (RAPD) analysis, or AP-PCR, is a novel DNA fingerprint technique [8,9] using single short primers of an arbitrary sequence to amplify genomic DNA in a low stringency PCR. The banding patterns obtained after electrophoresis of the PCR products have been used to fingerprint strains of various species [lo-141. Enterobacterial repetitive intergenic consensus (ERIC) sequence is a short interspersed repetitive nucleic-acid sequence originally found in Escherichia coli and S. typhimuiium. The use of outwardfacing primers complementary to each end of these repeats in a PCR has been described and termed ERIC-PCR [15]. This technique has recently proved
Microbiological Societies. Published by Elsevier Science B.V.

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Yue Millemann et al./ FEM.7 Immunology and Medical Microbiology 14 (1996) 129-134

to be useful in epidemiological studies of Enterobacter aerogenes and Escherichia coli isolates [16,17]. On Salmonella strains, only two studies using these latter methods are available [6,7], therefore the present study was undertaken on 70 strains of S. typhimurium and S. enteritidis isolated in poultryfarm animals to investigate the efficiency of both methods to differentiate field isolates. These animals were reared in different neighbouring flocks located in three regions. In such conditions, the study of clonal diffusion was of particular epidemiological interest. To assess the in vivo stability of RAPD and ERIC-PCR fingerprints, clonal strains regularly recovered from monoxenic animals were studied.

Mannheim, Germany), 5 pmol primer, 0.5 U Taq DNA Polymerase (Appligene, Illkirch, France), 10 ng of genomic DNA, in the recommended reaction buffer containing 10 mM Tris-HCl, 50 mM KCl, 1.5 n-&I MgCl,, 0.1% Triton 100X and 0.2 mg/ml gelatin. Amplifications were carried out in a PTC 100 thermal cycler (MJ Research, USA). Conditions for RAPD were as described previously [12]. Amplification products were separated by electrophoresis, through 1.5% agarose gels. The Raoul marker (Appligbne) was used as molecular weight standard. Reproducibility was tested by successive runs with the same samples or with different samples of DNA extracted from a single strain. Stability was tested on the isolates from monoxenic chickens. 2.3. ERIC-PCR 12.5 pmol of both primers ERIClR (5-ATGTAAGCTCCTGGGGATTCAC-3) and ERIC2 (5-AAGTAAGTGACTGGGGTGAGCG-3) [ 151 were added to the reaction volume. The amplification conditions (dNTP concentration, temperature and duration of extension, DNA concentration) were tested in order to obtain a good reproducibility of banding patterns. ERIC-PCR products were analysed after electrophoresis in 1% agarose gels. Stability was tested in similar conditions as for RAPD.

2. Materials and methods

2.1. Bacterial strains and DNA extraction

Fifty-six S. typhimurium and 14 S. enteritidis strains isolated between 1991 and 1993 in France from several avian sources in three independent geographical areas ((Y, p, 7) were studied [4]. The 56 S. typhimurium isolates were recovered from two avian species reared in 15 poultry farms. These strains were already characterized by ribotyping and IS200 typing. The S. enteritidis strains were isolated from three avian species reared in four poultry farms. We could not detect any difference among them by ribotyping or IS200 typing. In order to test the stability of RAPD types and ERIC-PCR fingerprints, S. typhimurium strain 183Fc1, from our collection, was inoculated to ten &day-old germ-free chickens, hatched and reared in sterile isolators for 15 weeks. The chickens were given sterile food and sterile water ad libitum. Bacterial clones were isolated every nine days from faeces and twelve of them were analysed and compared with each other and with the parental strain. DNA was isolated by the method of Wilson [ 181. 2.2. RAPD technique

3. Results and discussion Despite good sensitivity and reproducibility, the current genotypic methods used for typing Salmonella strains have some disadvantages. DNA hybridisation allows a clear interpretation, but the method is cumbersome, time-consuming, and requires appreciable amounts of DNA. However, since ribotyping has already been extensively used successfully for epidemiological studies, we stated ribotyping as a reference for a comparative study of the two techniques RAPD and ERIC-PCR. 3.1. RAPD analysis

The forty lo-mer primers from the G and H kits (Operon Technologies, USA) were evaluated. The following reagents were added in a 25 ~1 reaction volume and overlaid with mineral oil (Sigma Chemical, USA): 200 PM each nucleotide (Boehringer-

Preliminary studies were realized with the 40 primers on three strains of each serotype. From these results, five primers were retained for subsequent studies because of the clear and distinct banding

Ive Millemann et al./ FEMS Immunology and Medical Microbiology 14 (1996) 129-134

131

patterns obtained: 0PG04 (T-AGCGTGTCTG-31, OPG08 (S-TCACGTCCAC-3), OPGlO (S-AGGGCCGTCT-3), 0PH04 (S-GGAAGTCGCC-3) and 0PH13 (5-GACGCCACAC-3). Reproducibility of RAPD fingerprints was confirmed whatever the primer. In subsequent studies, only one RAPD profile was obtained with OPGO8 and 0PH13 for the 56 S. typhimurium strains studied. Three primers, OPG04, OPGlO and OPH04 allowed discrimination among them, leading to the definition of seven strain types characterized by their numerical codes (Table 1). RAPD profiles with OPH04 and 0PG04 differed only by the presence/absence of one and two bands, respectively. Patterns with OPGlO consisted in 5 to 9 bands ranging from I. to 8 kb approx. (Fig. 1A). From these patterns, tlhe strains from place cr could be discriminated from the strains isolated in places p and y. Six strains isolated from place p: BN91C3, BN92F1, BN92G3, BN92H2-BN92H4, shared OPGO4- and OPHO4pattems 1 and 9 respectively, with the ten strains isolated from place (Y (Table 1) and were thus discriminated from the other isolates
Table 1 PCR-types

from place 0. Thirty-three strains belonged to the major RAPD type 2-5-10. Other types were less represented. Comparison of discrimination obtained by RAPD assays with previous results of ribotyping [4] is presented in Table 2. The major RAPD fingerprint 2-5-10 was found among the strains of ribotypes 1, 2 and 5, and most of the strains of ribotypes 3, 6 and 7. RAPD fingerprint 2-7-10 grouped strains of ribotypes 4 and 8. RAPD fingerprint l-4-9 corresponded precisely to ribotype 9. Strains from ribotype 6 were subdivided into five different RAPD fingerprints. RAPD assays afforded separation between S. typhimurium which did not exactly correspond to results of ribotyping, and a better discrimination corroborated by the field indications of isolation was obtained with ribotyping. This is in contrast with the findings of other workers which have demonstrated results of RAPD and ribotyping being either in complete concordance for the study of Proteus mirabilis isolates [ 131, or at a very high level of concordance for Legionella pneumophila strains [14]. In a study on 56 Escherichia coli isolates, Alos et al. [lo]

of the 70 Salmonella strains 0PG04 pattern BN91A.5, BN91B1, 1 1 1 1 2 OPGlO pattern 4 4 8 5 5 OPH04 pattern 9 9 9 9 10 RAPDtype l-4-9 l-4-9 l-8-9 l-5-9 2-5-10 ERIC-PCR type II I I I I

S. typhimurium BN91A2, BN91A4, BN91B3-BN91B5

BN91A1, BN91A3, BN91B2 BN91C3 BN92F1, BN92G3, BN92H2-BN92H4 BN91DE, BN91C1, BN91C2, BN91C4, BN92C8, BN92D1, BN92D2, BN92ElBN92E3, BN92F2-BN92F4, BN92G1, BN92G2, BN92G4-BN93Gl1, BN91H1, BN92Kl-BN93K6, BN92M[l, BN92N1, BN93Pl BN91C7 BN91C6, BN92Jl-BN92J3, BN91C5 S. enteritidis BN92R1, BN92Sl-BN92S3, BN93T5, BN93T6, BN93U1, BN93U2 BN92R2 BN92R3, BN93Tl-BN93T4 BN93Ll

2 2 3 OPG08 pattern a b b

6 7 5 OPH 13 pattern
C

10 10 10 RAPD-type a-c b-d b-c

2-6-10 2-7-10 3-5-10 ERIC-PCR II II II type

I I I

d
C

Each strain symbol is composed of year of isolation (e.g. 91 for 1991), flock (A-U) and chronological Places of isolation were: (Y (flocks A, B; R and S); p (flocks C to L and T; IJ); y (flocks M to P).

order of isolation (l-1 1).

Table 2 Comparison of the results obtained by ribotyping and RAPD assays on 56 aviau S. fyphimurium strains

Ribotype

Strains with the following RAPD type

Total number of strains l-8-9 2-5-10 2-6-10 2-7-10 3-5-10 3 10 13 BN92Jl-BN92J3

l-4-9

l-5-9

b % \ 2 5 p

1 2 BN92G3 BN91DE, BN92C8, BN92G1, BN92G2, BN92G4-BN93Gll

BN92El-BN92E3 BN92Dl-BN92D2, BN92F3, BN92Kl-BN92K6, BN92Ml

s
% B

4 5 6 BN92H2BN92H4 BN92Fl BN92F2 BN9lC3 BN93Nl BN91Cl-BN91C2, BN91C4, BN92F4, BN91H1, BN93Pl

3 1 BN91C7 BN91C5 12

5 B B

7 8 9 5 1 33

BN91C6, BN93Ll

BN91Al-BN91A5, BN91Bl-BN91B5 1 5 1

2 2 10 56

z 3 B % B 2 % g

Total number

10

Yue Millemann et al. / FEMS Immunology and Medical Microbiology

14 (1996) 129-134

133

7378 2938 1810 1255 754554375 -

bp

23

45

Fig. 1. (A)RAPD profiles of strains of S. typhimurium with primer OPGlO (lanes l-5) ;md S. enteritidis with primers OPGOS (lanes 6 and 7) and OPH13 (lanes 8 and 9). Lanes 1-5: strains BN91A1, BN91DE, BN91C3, BN91C7, BN91C6; lanes 6-9: strains BN93T3, BN93T5, BN92R1, BN92R2. (B)ERIC-PCR profiles of strains of S. typhimurium (lanes l-3) and of S. enteritidis (lanes 4 and 5). Lanes l-5: strains BN91A1, BN91A2, BN91A3, BN92R1, BN92.S I. M: molecular weight marker Raoul (Appligbe, France).

reported concordant results although the discrimination with both methods was not exactly superimposable. Two other studies have also shown discrepancies between ribotyping and RAPD in the separation of E. coli strains [ 1!1,12]. The concomitant use of both methods could thus be recommended. In our study, the RAPD technique can only appear as a first approach for S. typhimurium isolates and should be completed by ribotyping, that remains the reference technique. Among the 14 S. enteritidis isolates, no discrimination could be ob:served with primers 0PG04, OPGlO and 0PH04. These strains were differentiated by the presence or absence of one band of approx. 385 bp with OPG08 and of three bands of approx. 800 bp, 1.1 kb and 1.9 kb with OPH13 (Fig. 1A). Thus, RAPD analysis separated the strains of S.

enteritidis into three groups (Table 11, in accordance with their origins and phenotypical characteristics. In flock T, the four strains BN93Tl-BN93T4 isolated from ducklings could be differentiated from strains BN93T5 and BN93T6 isolated later from adults. Moreover, the four strains BN93Tl-BN93T4 harboured three plasmids of 54, 50 and 3.8 kb and were resistant to quinolones, while strains BN93T5BN93T6 harboured only one plasmid of 54 kb and were susceptible to quinolones. In addition, phage typing (F. Grimont, personal communication) revealed that BN93T5 belonged to the common phage type 33 (phage typing scheme from the Institut Pasteur, France) as BN93Tl presented a more rarely encountered phage type 88a. Although our former results of ribotyping concluded in the presence of a unique clonal strain, from all these results, the presence of two different clonal strains in this particular flock can thus be hypothesized. The presence of three strains with different RAPD types in flock R could suggest independent contaminations possibly due to a lack of sanitary protection. To our knowledge, only one study has recently shown discrimination by RAPD assays, among avian and human isolates of S. enteritidis sharing the same phage type [6]. A single 15-mer oligonucleotide detected seven patterns among 35 unrelated isolates. However this discrimination was different from results of phage typing. Those seven amplification patterns were quite close to each other with a limited number of discriminative bands. A similar result can be underlined in our study with very close patterns among our strains. In our work, RAPD analysis appears as a method of interest which improves the tracing of avian S. enteritidis strains. 3.2. ERIC-PCR After preliminary assays of ERIC-PCR on our samples, optimized conditions were as defined: dNTP 0.2 mM, 1 min 30 only extension at 72C 40 ng DNA/reaction. Two stable and reproducible fingerprints were observed among the S. typhimurium and only one among the S. enteritidis strains (Fig. 1B). The fingerprints I and II were differentiated by the presence or absence of one band of approx. 1 kb. The fingerprint I was shared by the 43 S. typhimurium strains isolated from place p, three strains from place (Y and three strains from place y. The

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et al./ FEMS Immunology and Medical Microbiology

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fingerprint II was shared by the 7 last strains of S. and by all the strains of S. enteritidis. This is in disagreement with results of Van Lith and Aarts [7] which suggested a serotype-specific fingerprint. Our results suggest little core in this approach for epidemiological studies of SuZmoneEZa.
typhimurium 3.3. In vivo stability of RAPD types and ERIC-PCR profiles No variation was detected among the twelve clones collected in monoxenic animals during a 15 week experiment, which mimics the conventional time of rearing of chickens. Only one RAPD type and one ERIC-PCR fingerprint were found, identical respectively to the profiles of the parental strain. This confirms the in vivo stability of the RAPD types and ERIC-PCR fingerprints of Salmonella.

In conclusion, we find that ERIC-PCR is not appropriate for epidemiological studies of Salmonella strains in field situations. RAPD analysis seems well suited to such studies of S. enteritidis isolates where ribotyping is of limited value. For epidemiological studies of S. typhimurium ribotyping is still the most appropriate method.

Acknowledgements We are very grateful to A. BrCe and C. Mouline for skilled technical assistance, and to J.-F. Humbert for helpful discussion. We wish to thank F. Grimont for kindly having phage-typed strains BN93Tl and BN93T5 of our collection.

References
[l] Buisson, Y. (1992) La toxi-infection alirnentaire. Med. Mal. Infect. 22 (Special), 272-281. [2] Barker, R.M. (1980) Colicinogeny in Salmonella typhimurium. J. Gen. Microbial. 120, 21-26. [3] Threlfall, E.J., Hampton, M.D., Chart, H.. and Rowe, B. (1994) Use of plasmid profile typing for surveillance of Salmonella enferitidis phage type 4 from humans, poultry and eggs. Epidemlol. Infect. 112, 25-31. [4] Millemarm, Y., Lesage, MC., Chaslus-Dancla, E. and Lafont, J.P. (1995) Value of plasmid profiling, ribotyping and detection of IS200 for tracing avian isolates of Salmonella fyphimurium and enreritidis. J. Clin. Microbial. 33, 173-179.

[5] Olsen, J.E., Skov, M.N., Threlfall, E.J. and Brown, D.J. (1994) Clonal lines of Salmonella enterica serotype Enteritidis documented by IS200-, ribo-, pulsed-field gel electrophoresis and RFLP typing. J. Med. Microbial. 40, 15-22. [6] Fadl, A.A., Nguyen, A.V. and Khan, M.I. (1995) Analysis of Salmonella enteritidis isolates by arbitrarily primed PCR. J. Clin. Microbial. 33, 987-989. [7] Van Lith, L.A.J.T. and Aarts, H.J.M. (1994) Polymerase chain reaction identification of Salmonella serotypes. Lett. Appl. Microbial. 19, 273-276 [8] Welsh, J. and McClelland, M. (1990) Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 18, 7213-7218. [9] Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A. and Tingey, S.V. (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18, 6531-6535. [lo] Alos, J.I., Lambert, T. and Courvalin, P. (1993) Comparison of two molecular methods for tracing nosocomial transmission of Escherichia cali Kl in a neonatal unit. J. Clin. Microbial. 3 1, 1704-1709. [l I] Cave, H., Bingen, E., Elion, J. and Denamur, E. (1994) Differentiation of Escherichia coli strains using randomly amplified polymorphic DNA analysis. Res. Microbial. 145, 141-150. [12] Leroy-S&in, S., Lesage, M.C., Chaslus-Dancla, E. and Lafont, J.P. (1995) Clonal diffusion of EPEC-like Escherichia coli from rabbits as detected by ribotyping and random amplified polymorphic DNA assays. Epidemiol. Infect. 114, 113-121. [13] Bingen, E., Boissinot, C., Desjardins, P., Cave, H., Brahimi, N., Lambert-Zechovsky, N., Denamur, E., Blot, P. and Elion, J. (1993) Arbitrarily primed polymerase chain reaction provides rapid differentiation of Proteus mirabilis isolates from a pediatric hospital. J. Clin. Microbial. 31, 1055-1059. [14] Gomez-Lus, P., Fields, B.S., Benson, R.F., Martin, W.T., OConnor, S.P. and Black, CM. (1993) Comparison of arbitrarily primed polymerase chain reaction, ribotyping, and monoclonal antibody analysis for subtyping Legionella pneumophila serogroupl J. Clin. Microbial. 3 1, 1940- 1942. [15] Versalovic, J., Koeuth, T. and Lupski, J.R. (1991) Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res. 19, 6823-6831. [16] Georghiou, P.R., Hamill, R.J., Wright, C.E., Versalovic, J., Koeuth, T., Watson, D.A. and Lupski, J.R. (1995) Molecular epidemiology of infections due to Enterobacter aerogenes: identification of hospital-associated strains by molecular techniques. Clin. Infect. Dis. 20, 84-94. [17] Lipman, L.J.A., de Nijs A., Lam, T.J.G.M. and Gaastra W. (1995) Identification of Escherichia coli strains from cows with clinical mastitis by serotyping and DNA polymorphism patterns with REP and ERIC primers. Vet. Microbial. 43, 13-19. [18] Wilson, K. (1987) Preparation of genomic DNA from bacteria. In: Current Protocols in Molecular Biology (Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K., Eds.), pp. 2.4.1.-.2.4.2. Wiley, New York.