Comp RAPD-ERIC 4 Salm

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

Comparison of random amplified polymorphic DNA analysis

IMMUNOLOGY NDMEDIC LMICROBIOLOGY

and enterobacterial repetitive intergenic consensus-PCRfor 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, Centre de Tours-Nouzilly,37380 Monnaie, France

Received 16 January 1996; revised 22 February 1996; accepted 22 February 1996

AbstractDiscrimination of 70 Salmonella trains previously studied by ribotyping was realized by RAPD and ERIC-PCR analysis.

RAPD results on the 56 S typhimur iumsolates did not closely match those of ribotyping. With ERIC-PCR, two fingerprintsonly were obtained. For the 14 S. enteritidis trains, a helpful discrimination was obtained with RAPD analysis, whileERIC-PCR resulted in a single fingerprint.Keywords: RAPD; ERIC-PCR; Ribotyping; Epidemiology; Salmonella typhimurium; Salmonella enteritidis

1 IntroductionSalmonella typhimurium and S. enteritidis are

among the major etiologic agents of human gastro-enteritis in France [l] and are also the serotypes mostfrequently isolated frolm poultry products. Epidemio-logical studies of Salmonella infections require theuse of efficient molecular markers to trace preciselythe diffusion of strains. Phenotypic and genotypictechniques have been used for subtyping Salmonellaserotypes, including phage and bacteriocin typing,plasmid profile analysis, RFLP, ribotyping, IS200

typing, pulsed-field gel electrophoresis, and recentlyarbitrarily primed polymerase chain reaction (AP-PCR) and ERIC-PCR [2-71.Random Amplified Polymorphic DNA (RAPD)analysis, or AP-PCR, is a novel DNA fingerprinttechnique [8,9] using single short primers of anarbitrary sequence to amplify genomic DNA in a lowstringency PCR. The banding patterns obtained afterelectrophoresis of the PCR products have been usedto fingerprint strains of various species [lo-141.Enterobacterial repetitive intergenic consensus(ERIC) sequence is a short interspersed repetitivenucleic-acid sequence originally found in Escher-ichia coli and S. ty phimuii um. The use of outward-facing primers complementary to each end of theserepeats in a PCR has been described and termedERIC-PCR [15]. This technique has recently proved

* Corresponding author. Tel: +33 47 42 77 65; Fax: +33 4742 77 74; E-mail: [email protected] Present address: Patholoaie du B&ail. DPASA. Ecole Na-tionale V&%inaire dAlfort, 94704 Maisons-Alfort Cedex, France.09288244/96/$15.00 Copyright 0 1996 Federation of European Microbiological Societies. Published by Elsevier Science B.V.PZI SO928-8244(96)00021-l


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13 YueM il lemann et al./ FEM.7 Immunology and M edical M icrobiol ogy 14 1996) 129-134

to be useful in epidemiological studies of Enterobac-ter aerogenes and Escherichia coli isolates [16,17].

On Salmonella strains, only two studies usingthese latter methods are available [6,7], therefore thepresent study was undertaken on 70 strains of S.typhimurium and S. enteritidis isolated in poultry-farm animals to investigate the efficiency of bothmethods to differentiate field isolates. These animalswere reared in different neighbouring flocks locatedin three regions. In such conditions, the study ofclonal diffusion was of particular epidemiologicalinterest. To assess the in vivo stability of RAPD andERIC-PCR fingerprints, clonal strains regularly re-covered from monoxenic animals were studied.

Mannheim, Germany), 5 pmol primer, 0.5 U TaqDNA Polymerase (Appligene, Illkirch, France), 10ng of genomic DNA, in the recommended reactionbuffer containing 10 mM Tris-HCl, 50 mM KCl, 1.5n-&I MgCl,, 0.1% Triton 100X and 0.2 mg/mlgelatin. Amplifications were carried out in a PTC100 thermal cycler (MJ Research, USA). Conditionsfor RAPD were as described previously [12]. Ampli-fication 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 withthe same samples or with different samples of DNAextracted from a single strain. Stability was tested onthe isolates from monoxenic chickens.

2. Materials and methods 2.3. ERIC-PCR2 1 Bacterial strains and DNA extraction

Fifty-six S. typhimurium and 14 S. enteritidisstrains isolated between 1991 and 1993 in Francefrom several avian sources in three independent geo-graphical areas ((Y, p, 7) were studied [4]. The 56S. typhimurium isolates were recovered from twoavian species reared in 15 poultry farms. Thesestrains were already characterized by ribotyping andIS200 typing. The S. enteritidis strains were isolatedfrom three avian species reared in four poultry farms.We could not detect any difference among them byribotyping or IS200 typing.

12.5 pmol of both primers ERIClR (5-ATGTAA-GCTCCTGGGGATTCAC-3) and ERIC2 (5-AAG-TAAGTGACTGGGGTGAGCG-3) [ 151 were addedto the reaction volume. The amplification conditions(dNTP concentration, temperature and duration ofextension, DNA concentration) were tested in orderto obtain a good reproducibility of banding patterns.ERIC-PCR products were analysed after elec-trophoresis in 1% agarose gels. Stability was testedin similar conditions as for RAPD.

3. Results and discussionIn order to test the stability of RAPD types and

ERIC-PCR fingerprints, S. typhimurium strain183Fc1, from our collection, was inoculated to ten&day-old germ-free chickens, hatched and reared insterile isolators for 15 weeks. The chickens weregiven sterile food and sterile water ad libitum. Bacte-rial clones were isolated every nine days from faecesand twelve of them were analysed and comparedwith each other and with the parental strain.

DNA was isolated by the method of Wilson [ 181.

Despite good sensitivity and reproducibility, thecurrent genotypic methods used for typing Salmon-ella strains have some disadvantages. DNA hybridis-ation allows a clear interpretation, but the method iscumbersome, time-consuming, and requires appre-ciable amounts of DNA. However, since ribotypinghas already been extensively used successfully forepidemiological studies, we stated ribotyping as areference for a comparative study of the two tech-niques RAPD and ERIC-PCR.2.2. RAPD technique3.1. RAPD analysisThe forty lo-mer primers from the G and H kits

(Operon Technologies, USA) were evaluated. Thefollowing reagents were added in a 25 ~1 reactionvolume and overlaid with mineral oil (Sigma Chemi-cal, USA): 200 PM each nucleotide (Boehringer-

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


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I ve M il lemann et al./ FEMS Immunology and M edical M icrobiol ogy 14 1996) 129-134 131

patterns obtained: 0PG04 (T-AGCGTGTCTG-31,OPG08 (S-TCACGTCCAC-3), OPGlO (S-AGG-GCCGTCT-3), 0PH04 (S-GGAAGTCGCC-3) and0PH13 (5-GACGCCACAC-3). Reproducibility ofRAPD fingerprints was confirmed whatever theprimer.

from place 0. Thirty-three strains belonged to themajor RAPD type 2-5-10. Other types were lessrepresented.

In subsequent studies, only one RAPD profile wasobtained with OPGO8 and 0PH13 for the 56 S.typhimurium strains studied. Three primers, OPG04,OPGlO and OPH04 allowed discrimination amongthem, leading to the definition of seven strain typescharacterized by their numerical codes (Table 1).RAPD profiles with OPH04 and 0PG04 differedonly by the presence/absence of one and two bands,respectively. Patterns with OPGlO consisted in 5 to 9bands ranging from I. to 8 kb approx. (Fig. 1A).From these patterns, tlhe strains from place cr couldbe discriminated from the strains isolated in places pand y. Six strains isolated from place p: BN91C3,BN92F1, BN92G3, BN92H2-BN92H4, sharedOPGO4- and OPHO4pattems 1 and 9 respectively,with the ten strains isolated from place (Y (Table 1)and were thus discriminated from the other isolates

Comparison of discrimination obtained by RAPDassays with previous results of ribotyping [4] ispresented in Table 2. The major RAPD fingerprint2-5-10 was found among the strains of ribotypes 1, 2and 5, and most of the strains of ribotypes 3, 6 and7. RAPD fingerprint 2-7-10 grouped strains of ribo-types 4 and 8. RAPD fingerprint l-4-9 correspondedprecisely to ribotype 9. Strains from ribotype 6 weresubdivided into five different RAPD fingerprints.RAPD assays afforded separation between S. ty-phimurium which did not exactly correspond to re-sults of ribotyping, and a better discrimination cor-roborated by the field indications of isolation wasobtained with ribotyping. This is in contrast with thefindings of other workers which have demonstratedresults of RAPD and ribotyping being either in com-plete concordance for the study of Proteus mirabilisisolates [ 131, or at a very high level of concordancefor Legionella pneumophila strains [14]. In a studyon 56 Escherichia coli isolates, Alos et al. [lo]

Table 1PCR-types of the 70 Salmonella strainsS. ty phimurium 0PG04 OPGlO OPH04 RAPD- ERIC-PCR

pattern pattern pattern type typeBN91A2, BN91A4, BN91A.5, BN91B1, 1 4 9 l-4-9 IIBN91B3-BN91B5BN91A1, BN91A3, BN91B2 1 4 9 l-4-9 IBN91C3 1 8 9 l-8-9 IBN92F1, BN92G3, BN92H2-BN92H4 1 5 9 l-5-9 IBN91DE, BN91C1, BN91C2, BN91C4, 2 5 10 2-5-10 IBN92C8, BN92D1, BN92D2, BN92El-BN92E3, BN92F2-BN92F4, BN92G1,BN92G2, BN92G4-BN93Gl1, BN91H1,BN92Kl-BN93K6, BN92M[l, BN92N1,BN93PlBN91C7 2 6 10 2-6-10 IBN91C6, BN92Jl-BN92J3, BN93Ll 2 7 10 2-7-10 IBN91C5 3 5 10 3-5-10 IS. enterit idi sBN92R1, BN92Sl-BN92S3, BN93T5,

OPG08 pattern OPH 13 pattern RAPD-type ERIC-PCR typea a-c II

BN93T6, BN93U1, BN93U2BN92R2BN92R3, BN93Tl-BN93T4

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Each strain symbol is composed of year of isolation (e.g. 91 for 1991), flock (A-U) and chronological order of isolation (l-1 1).Places of isolation were: (Y (flocks A, B; R and S); p (flocks C to L and T; IJ); y (flocks M to P).


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Yue Millemann et al. / FEMS Immunology and Medical Microbiology 14 1996) 129-134 133

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Fig. 1. A) APD profiles of strains of S. typhimurium withprimer OPGlO (lanes l-5) ;md S. enteritidis with primers OPGOS(lanes 6 and 7) and OPH13 (lanes 8 and 9). Lanes 1-5: strainsBN91A1, BN91DE, BN91C3, BN91C7, BN91C6; lanes 6-9:strains BN93T3, BN93T5, BN92R1, BN92R2. B) RIC-PCRprofiles 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 discrimina-tion with both methods was not exactly superimpos-able. Two other studies have also shown discrepan-cies between ribotyping and RAPD in the separationof E. coli strains [ 1 1,12]. The concomitant use ofboth methods could thus be recommended. In ourstudy, the RAPD technique can only appear as a firstapproach for S. typhimurium isolates and should becompleted by ribotyping, that remains the referencetechnique.Among the 14 S. enteritidis isolates, no discrimi-nation could be ob:served with primers 0PG04,OPGlO and 0PH04. These strains were differenti-ated by the presence or absence of one band ofapprox. 385 bp with OPG08 and of three bands ofapprox. 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 accordancewith their origins and phenotypical characteristics. Inflock T, the four strains BN93Tl-BN93T4 isolatedfrom ducklings could be differentiated from strainsBN93T5 and BN93T6 isolated later from adults.Moreover, the four strains BN93Tl-BN93T4 har-boured three plasmids of 54, 50 and 3.8 kb and wereresistant to quinolones, while strains BN93T5-BN93T6 harboured only one plasmid of 54 kb andwere susceptible to quinolones. In addition, phagetyping (F. Grimont, personal communication) re-vealed that BN93T5 belonged to the common phagetype 33 (phage typing scheme from the Institut Pas-teur, France) as BN93Tl presented a more rarelyencountered phage type 88a. Although our formerresults of ribotyping concluded in the presence of aunique clonal strain, from all these results, the pres-ence of two different clonal strains in this particularflock can thus be hypothesized. The presence ofthree strains with different RAPD types in flock Rcould suggest independent contaminations possiblydue to a lack of sanitary protection.To our knowledge, only one study has recentlyshown discrimination by RAPD assays, among avianand human isolates of S. enteritidis sharing the samephage type [6]. A single 15-mer oligonucleotide de-tected seven patterns among 35 unrelated isolates.However this discrimination was different from re-sults of phage typing. Those seven amplificationpatterns were quite close to each other with a limitednumber of discriminative bands. A similar result canbe underlined in our study with very close patternsamong our strains. In our work, RAPD analysisappears as a method of interest which improves thetracing of avian S. enteritidis strains.3.2. ERIC-PCRAfter preliminary assays of ERIC-PCR on oursamples, optimized conditions were as defined: dNTP0.2 mM, 1 min 30 only extension at 72C 40 ngDNA/reaction. Two stable and reproducible finger-prints were observed among the S. typhimurium andonly one among the S. enteritidis strains (Fig. 1B).The fingerprints I and II were differentiated by thepresence or absence of one band of approx. 1 kb.The fingerprint I was shared by the 43 S. typhi-murium strains isolated from place p, three strainsfrom place (Y and three strains from place y. The


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134 YueMillenann et al./ FEMS Immunology and Medical Microbiology I4 1996) 129-134

fingerprint II was shared by the 7 last strains of S.typhimurium and by all the strains of S. enteritidis.This is in disagreement with results of Van Lith andAarts [7] which suggested a serotype-specific finger-print. Our results suggest little core in this approachfor epidemiological studies of SuZmoneEZa.3.3. In vivo stability of RAPD types and ERIC-PCRprofiles

No variation was detected among the twelveclones collected in monoxenic animals during a 15week experiment, which mimics the conventionaltime of rearing of chickens. Only one RAPD typeand one ERIC-PCR fingerprint were found, identicalrespectively to the profiles of the parental strain.This confirms the in vivo stability of the RAPDtypes and ERIC-PCR fingerprints of Salmonella.In conclusion, we find that ERIC-PCR is not

appropriate for epidemiological studies of Salmonellastrains in field situations. RAPD analysis seems wellsuited to such studies of S. enteritidis isolates whereribotyping is of limited value. For epidemiologicalstudies of S. typhimurium ribotyping is still the mostappropriate method.

AcknowledgementsWe are very grateful to A. BrCe and C. Moulinefor skilled technical assistance, and to J.-F. Humbert

for helpful discussion. We wish to thank F. Grimontfor kindly having phage-typed strains BN93Tl andBN93T5 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 ty-

phimurium. 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 ofSalmonella enferitidis phage type 4 from humans, poultryand eggs. Epidemlol. Infect. 112, 25-31.

[4] Millemarm, Y., Lesage, MC., Chaslus-Dancla, E. and La-font, J.P. (1995) Value of plasmid profiling, ribotyping anddetection of IS200 for tracing avian isolates of Salmonellafyphimurium 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 Enteri-tidis documented by IS200-, ribo-, pulsed-field gel elec-trophoresis and RFLP typing. J. Med. Microbial. 40, 15-22.[6] Fadl, A.A., Nguyen, A.V. and Khan, M.I. (1995) Analysis ofSalmonella 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) Polymerasechain reaction identification of Salmonella serotypes. Lett.Appl. Microbial. 19, 273-276

[8] Welsh, J. and McClelland, M. (1990) Fingerprinting genomesusing 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 byarbitrary primers are useful as genetic markers. NucleicAcids Res. 18, 6531-6535.[lo] Alos, J.I., Lambert, T. and Courvalin, P. (1993) Comparisonof two molecular methods for tracing nosocomial transmis-sion 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 randomlyamplified polymorphic DNA analysis. Res. Microbial. 145,141-150.

[12] Leroy-S&in, S., Lesage, M.C., Chaslus-Dancla, E. and La-font, J.P. (1995) Clonal diffusion of EPEC-like Escherichiacoli from rabbits as detected by ribotyping and randomamplified 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 pro-vides rapid differentiation of Proteus mirabilis isolates froma 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 ofarbitrarily primed polymerase chain reaction, ribotyping, andmonoclonal antibody analysis for subtyping Legionellapneumophila serogroupl J. Clin. Microbial. 3 1, 1940- 1942.

[15] Versalovic, J., Koeuth, T. and Lupski, J.R. (1991) Distribu-tion of repetitive DNA sequences in eubacteria and applica-tion to fingerprinting of bacterial genomes. Nucleic AcidsRes. 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) Molecularepidemiology of infections due to Enterobacter aerogenes:identification of hospital-associated strains by moleculartechniques. 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 cowswith clinical mastitis by serotyping and DNA polymorphismpatterns with REP and ERIC primers. Vet. Microbial. 43,13-19.

[18] Wilson, K. (1987) Preparation of genomic DNA from bacte-ria. 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.

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