Value of Plasmid Profiling, Ribotyping, and Detection of IS200 for

JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1995, p. 173–179 0095-1137/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 33, No. 1

Value of Plasmid Profiling, Ribotyping, and Detection of IS200 for Tracing Avian Isolates of Salmonella typhimurium and S. enteritidis YVES MILLEMANN,† MARIE-CLAUDE LESAGE, ELISABETH CHASLUS-DANCLA,* AND JEAN-PIERRE LAFONT Station de Pathologie Aviaire et Parasitologie, Institut National de la Recherche Agronomique, Centre de Tours-Nouzilly, 37380 Monnaie, France Received 13 May 1994/Returned for modification 4 August 1994/Accepted 11 October 1994

Seventy selected strains of Salmonella typhimurium and S. enteritidis isolated from related poultry flocks in three independent geographical areas were characterized by phenotypic and genotypic methods to compare the usefulness of the methods in epidemiological studies. The 56 S. typhimurium isolates were poorly discriminated by their biotypes, resistance patterns, and plasmid profiles. Nine different ribotypes were obtained after DNA digestion by BglII, PvuII, and SmaI. Seven IS200 types, characterized by six to nine copies of IS200 on the chromosome, were detected after digestion of genomic DNA by PstI. These studies resulted in the definition of 15 clonal lineages distributed in three clusters. The 14 S. enteritidis strains were not discriminated either by ribotyping or by detection of IS200 (IS200 typing), but were separated on the basis of antibiotic resistance and plasmid profiling. The stability of the insertion sequence type was confirmed by inoculation of an S. typhimurium strain to axenic chickens reared for 15 weeks in sterile isolators. chromosome of the great majority of Salmonella strains, and additional IS200 copies on plasmid DNA have recently been reported (24). Studies have already been carried out on the copy number and location of IS200 for collections of Salmonella strains of unrelated and related origins. They have pointed out the phylogenetic and epidemiological interest of the determination of IS200 types of Salmonella serovars of public health significance (15, 21–23). The purpose of the present investigation was to discriminate 70 strains of S. enteritidis and S. typhimurium isolated in poultry farms by phenotypic characterization, plasmid profiling, and chromosomal fingerprinting. We were interested in comparing the usefulness of ribotyping and detection of IS200 (IS200 typing) in such epidemiological studies on Salmonella strains, and we studied the stability of the IS types under controlled experimental conditions.

Salmonella spp. are the primary bacterial agents responsible for foodborne outbreaks of human gastrointestinal disease in France (5). Two serotypes are of particular clinical importance: S. enteritidis and S. typhimurium. These serovars have been predominantly isolated for several years in France, with an increasing rate of isolation for S. enteritidis. Poultry products are often suspected sources of infection of human gastroenteritis when S. enteritidis or S. typhimurium strains are isolated. It is of interest to have available molecular markers that can be used to trace clonal strains of these two serovars and to relate outbreaks to poultry sources. Such molecular markers are also of prominent usefulness in the poultry sector for epidemiological investigations aimed at finding the origins of the flock contamination. The analysis of phenotypic characteristics can be considered, as can plasmid content analysis (25, 27, 28). Chromosomal fingerprinting appears to be more adequate for the study of clonal diffusion. Endonuclease-cleaved chromosomal DNA can be hybridized with the universal probe 16S123S rRNA corresponding to highly conserved sequences (7, 10, 12, 18, 29). Insertion sequences (ISs) can be also used as probes. Because they correspond to DNA sequences subject to variations in copy numbers and locations, they can improve the analysis of chromosomal DNA digested by restriction endonucleases. Such ISs have been tested in some bacterial species: IS256 in Staphylococcus aureus and IS986 or IS6110 in Mycobacterium tuberculosis (6, 13, 30). IS200 has been identified in a mutant strain of S. typhimurium (11). This short IS appeared first to be specific to Salmonella spp., but some later studies have shown that other bacteria, like Escherichia coli or Shigella spp. (3), can harbor IS200 copies and that some Salmonella strains do not have any. However, IS200 copies can be detected on the

MATERIALS AND METHODS Bacterial strains. We studied 56 S. typhimurium and 14 S. enteritidis strains isolated from several avian sources between 1991 and 1993 in France, where recurrent problems of Salmonella contamination had been identified. The properties and origins of the strains are presented in Tables 1 and 2. The strains were isolated from animals reared in three independent geographical areas. In each place the different farmhouses were integrated into the same avian production system. From the first place, 10 S. typhimurium (strains BN91A1 to BN91B5 in Table 1) and 6 S. enteritidis strains (strains BN92R1 to BN92S3 in Table 2) were isolated from four different flocks (flocks A, B, R, and S). From the second place, 43 S. typhimurium strains (strains BN91C1 to BN93L1 in Table 1) and 6 S. enteritidis strains (strains BN93T1 to BN93T6 in Table 2) were isolated from ducks reared in nine different farmhouses (flocks C to L and T). S. typhimurium BN91DE was isolated from the parents of ducks reared in flocks D and E. From this place, two S. enteritidis strains (strains BN93U1 and BN93U2 in Table 2) were obtained from a diseased rabbit and were isolated before and after treatment with quinolones, respectively. Three S. typhimurium strains (strains BN92M1, BN93N1, and BN93P1) were isolated from ducks reared in three different farmhouses (flocks M, N, and P) located in the third place. S. typhimurium 183Fc1, from our collection, was used to test the stability of IS types when inoculated into young animals under very controlled conditions. Biotypes and resistance to antibiotics. All strains were biotyped by determining the results of 23 different biochemical tests with API 20E strips (BioMe´rieux, Marcy-L’Etoile, France). The strains were screened for their resistances to the following seven antibiotics (all of which were obtained from Sanofi-Diagnostics Pasteur, Marnesla-Coquette, France): ampicillin (10 mg), chloramphenicol (30 mg), kanamycin (30 mg), streptomycin (10 mg), tetracycline (30 mg), trimethoprim (5 mg), and

* Corresponding author. Mailing address: Station de Pathologie Aviaire et Parasitologie, Institut National de la Recherche Agronomique, Centre de Tours-Nouzilly, 37380 Monnaie, France. Phone: 3347-42-77-65. Fax: 33-47-42-77-74. Electronic mail address: Elisabeth. [email protected]. † Present address: Pathologie du Be´tail, DPASA, Ecole Nationale Ve´te´rinaire d’Alfort, 94704 Maisons-Alfort Cedex, France. 173

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J. CLIN. MICROBIOL. TABLE 1. Source and properties of the 56 S. typhimurium strains

a

Strain and flock

BN91A1 BN91A2 BN91A3 BN91A4 BN91A5 BN91B1 BN91B2 BN91B3 BN91B4, BN91B5 BN91C1 BN91C2, BN91C7 BN91C3, BN91C4 BN91C5 BN91C6 BN92C8 BN91DE BN92D1 BN92D2 BN92E1 BN92E2, BN92E3 BN92F1 BN92F2 BN92F3 BN92F4 BN92G1 BN92G2 BN92G3 BN92G4 BN93G5, BN93G6 BN93G7, BN93G11 BN93G8–BN93G10 BN91H1 BN92H2–BN92H4 BN92J1, BN92J3 BN92J2 BN92K1 BN92K2–BN92K4 BN93K5 BN93K6 BN93L1 BN92M1 BN93N1 BN93P1 a b c

Source

Turkey, litter Turkey, rearing house Turkey, rearing Turkey, egg Turkey, egg Turkey, rearing Turkey, meconium Turkey, water Turkey, nests Duck, parents Duck, rearing Duck, rearing Duck, rearing Duck, rearing Duck, egg Duck, parents Duck, rearing Duck, rearing Duck, rearing Duck, rearing Duck, rearing Duck, rearing Duck, rearing Duck, egg Duck, rearing Duck, rearing Duck, egg Duck, egg Duck, hatching house Duck, rearing Duck, egg Duck, rearing Duck, rearing Duck, rearing Duck, rearing Duck, hatching house Duck, rearing Duck, egg Duck, rearing Duck, food Duck, egg Duck, egg Duck, egg

Biochemical particularity b

— — — — — — — — — — — — — Inositol negative — Sorbitol negative Sorbitol negative — Sorbitol negative Sorbitol negative — — Sorbitol negative — — — Sodium citrate negative — Sorbitol negative Sorbitol negative Sorbitol negative — — Sorbitol negative — Sorbitol negative Sorbitol negative Sorbitol negative Sorbitol negative Inositol negative Sorbitol negative Sorbitol negative Sodium citrate negative

Antibiotic resistance c

— — — — Nal, Oxo, Pef — — — — — — — — Ap, Cm, Tc, Tp Cm, Tc — — — — — — — — — — — — — — — — — — Cm, Tc, Tp Cm, Tc, Tp — — — — — — Cm, Tc —

Plasmid size (kb)

90 90, 3.5 90 90, 3.5 90, 3.5 90, 3.5 90 90, 3.5 90, 3.5 90, 2.8, 2.5 90, 2.5 90, 2.8, 2.5 90, 40, 2.8, 2.5 115, 100, 40, 2.2, 2 90, 40 90 90 90 90 90, 40 90, 40, 2.8, 2.5 90, 2.8, 2.5 90 90, 40, 2.8, 2.5 90 90, 40 90 90, 40 90 90 90 90, 40, 2.8, 2.5 90, 40, 2.8, 2.5 100, 90 100, 90 90 90 90 90 90 90 90, 40 90, 3.5

The symbol for each strain is composed of year of isolation (e.g., 92 for 1992), flock (letter), and chronological order of isolation. —, none (API profile 670 047 52). —, susceptible. Ap, ampicillin; Cm, chloramphenicol; Nal, nalidixic acid; Oxo, oxolinic acid; Pef, pefloxacin; Tc, tetracycline; Tp, trimethoprim.

nalidixic acid (30 mg). The resistances were determined by the standardized disk diffusion technique (2). Strains resistant to nalidixic acid were subsequently tested for their resistances to oxolinic acid (10 mg), ciprofloxacin (5 mg), norfloxacin (5 mg), ofloxacin (5 mg), and pefloxacin (5 mg). Extraction and analysis of plasmid DNA. Isolation of plasmid DNA was performed as described by Takahashi and Nagano (26). Samples of plasmid DNA were electrophoresed in horizontal 0.8% agarose gels. The reference plasmids RP4 (54 kb) and R112 (100.5 kb) and the supercoiled DNA ladder (Gibco Bethesda Research Laboratories) were used as molecular markers. For further discrimination, plasmid DNA was digested to completion in 30-ml volumes with EcoRI or HindIII (Boehringer-Mannheim, Mannheim, Germany). Phage lambda DNA digested under the same conditions was used as a molecular weight standard. Genomic DNA preparation. Total DNA was extracted as described by Wilson (31). DNA (2 to 5 mg) was digested with 10 U of restriction endonucleases (Boehringer-Mannheim). Four different endonucleases were chosen for use in ribotyping after preliminary experiments, as follows: BglII, HindIII, PvuII, and SmaI. For IS typing, BglII, PvuII, PstI, and SmaI were used because of the lack of corresponding restriction sites within the IS sequence. Genomic restriction digests were electrophoresed on 0.8% horizontal agarose gels and were then vacuum blotted (LKB Pharmacia, Uppsala, Sweden) onto Hybond-N nylon membranes (Amersham International plc, Amersham, England). The Raoul marker (Applige`ne, Illkirch, France) was used as a molecular

weight standard, and the profiles were compared on a Sparc station with the BioImage System (Millipore). Labeling and hybridization of probes. For ribotyping, blots of digested electrophoresed DNA were hybridized with the ribosomal DNA (rDNA) probe obtained after the action of a reverse transcriptase on 16S123S rRNA isolated from E. coli by the method previously described by N. Picard-Pasquier et al. (17). For IS200 typing, plasmid pIZ46, which was kindly provided by Gibert et al. (9), was used to transform E. coli HB101 cells. Amplification, extraction, and purification in a cesium chloride gradient were performed as described previously (20). An internal 600-bp EcoRI fragment of pIZ46 corresponding to the dimer of the 300-bp EcoRI-HindIII fragment of IS200 was isolated by gel electrophoresis, eluted by an agarase extraction procedure (8), and used as the probe (9). Probes were radiolabeled with [a-32P]dCTP by using the Multiprime kit (Amersham). Hybridization was conducted at 658C in a hybridization oven (Applige`ne). The filters were stringently washed. Homologous bands were visualized by autoradiography on HyperfilmMP films (Amersham). Data analysis. rDNA patterns and IS200 types were analyzed, and the presence or absence of a band was coded as 1 or 0, respectively, in a data matrix. The Jaccard’s distance coefficient (D) between each pair of ribotypes or IS types was calculated as follows: D 5 1 2 [C/(2N 2 C)], where C is the number of bands in common and N is the total number of the different bands of each strain. Agglomerative hierarchical cluster analysis was derived from the distance

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TABLE 2. Source and properties of the 14 S. enteritidis strains Strain and flocka

BN92R1 BN92R2, BN92R3 BN92S1, BN92S2 BN92S3 BN93T1, BN93T3 BN93T2 BN93T4 BN93T5, BN93T6 BN93U1 BN93U2

Source

Antibiotic resistance

Guinea fowl, parents —b Guinea fowl, young — Duck, parents — 1-day-old duck — Duck, down Nal, Oxo Duck, meconium Nal Duck meconium Nal, Oxo Duck, rearing — Rabbit 1, ill — Rabbit 1, after treatment Nal, Oxo, Pef

Plasmid size (kb)

54 54 54 54 54, 50, 3.8 54, 50, 3.8 54, 50, 3.8 54 54 54

a The symbol for each strain is composed of year of isolation (e.g., 92 for 1992), flock (letter), and chronological order of isolation. b —, susceptible. Nal, nalidixic acid; Oxo, oxolinic acid; Pef, Pefloxacin.

matrices by using the unweighted pair-group method with arithmetic averages algorithm, and correspondence analysis was also conducted on the data matrix described previously; both of these analyses were conducted with the commercial software program StatITCF (Institut des Ce´re ´ales et des Fourrages, Paris, France). Stability of IS types in strains inoculated into animals under controlled conditions. Ten axenic White Leghorn chickens were hatched and reared in sterile isolators for 15 weeks. They were given sterile food and water ad libitum. When they were 8 days old, each animal received S. typhimurium 183Fc1 per os at an inoculum of 2 3 106 bacteria. The salmonellae were recovered every 9 days over a period of 15 weeks from the feces of the chickens on Salmonella-Shigella agar (Sanofi-Diagnostics Pasteur), and 12 independent colonies were studied and compared with each other and with the parental strain by plasmid profiling, ribotyping, and IS200 typing.

RESULTS Biotypes and resistance to antibiotics. The 56 S. typhimurium strains could be discriminated into four biotypes. Differences in the utilization of citrate and in the fermentation and oxidation of inositol and sorbitol were observed (Table 1). Only 1 among the 14 S. enteritidis strains, BN93U1, could be discriminated on the basis of utilization of amygdalin. Twelve of the 70 strains tested (17.1%) were resistant to antibiotics. Resistance to chloramphenicol and tetracycline

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was the most commonly encountered resistance in S. typhimurium strains. Four S. enteritidis strains were resistant to quinolones (Table 2). Plasmid content. The sizes of the plasmids present in the 70 strains are given in Tables 1 and 2. The molecular sizes ranged from 2 to 115 kb. All but one of the S. typhimurium isolates, isolate BN91C6, harbored a 90-kb plasmid which is thought to be the serotype-specific virulence plasmid (18). Eight different plasmid profiles were determined among the 56 S. typhimurium isolates. Strains from within each flock of flocks D, H, J, and K had the same plasmid profiles. A low level of diversity was observed in the plasmid profiles of strains isolated from flocks A, B, E, and G. These profiles were differentiated only by one or a limited number of plasmids, mainly of low molecular weight. Profiles for strains isolated from flocks C and F showed a larger degree of diversity. The three strains from flocks M, N, and P showed plasmid profiles already encountered among the other flocks. The plasmid restriction patterns confirmed these results (data not shown). All of the S. enteritidis strains harbored a 54-kb plasmid that probably corresponded to the serotype-specific virulence plasmid (19). Two plasmid profiles were detected among the 14 S. enteritidis strains isolated from flocks R to U. Four isolates harbored two more plasmids. The plasmid restriction fingerprints suggest the presence of a unique 54-kb plasmid. The plasmid profiles of the clonal strains in 15-week experiment were determined. No variation in the three plasmids of 90, 60, and 3 kb was observed. Only one plasmid restriction pattern was obtained (data not shown). Ribotyping. Nine different rDNA restriction patterns were defined among the 56 S. typhimurium isolates (Table 3; Fig. 1). Ribotypes 1 to 7 were closely related and only differed by restriction patterns with one enzyme. Ribotypes 8 and 9 were closely related and were clearly distinguishable from the former ribotypes. Ribotype 8 grouped two strains: one, BN91C6, isolated from flock C, and one, BN93L1, isolated from food. Ribotype 9 grouped the strains from flocks A and B, which were located in the same area. For strains isolated from flocks E, D, G, H, J, and K, the strains within each flock shared a similar ribotype. Strains isolated from flocks C and F were

TABLE 3. Ribotypes and IS200 types of avian isolates of S. typhimurium and S. enteritidis rDNA pattern Strain

S. typhimurium BN92E1, BN92E2 BN92E3 BN92D1, BN92D2, BN92K1–BN93K6, BN92F3 BN92M1 BN91DE, BN92C8, BN92G1–BN93G11 BN92J2 BN92J1, BN92J3 BN92N1 BN91C1, BN91C5, BN91C7, BN92F4, BN91H1, BN92H2, BN93P1 BN91C2–BN91C4, BN92H3, BN92H4 BN92F2 BN92F1 BN91C6, BN93L1 BN91A1, BN91A3–BN91A5, BN91B1–BN91B5 BN91A2 S. enteritidis BN92R1–BN93U2

IS200 type HindIII

BglII

PvuII

SmaI

Ribotype no.

H1 H1 H1 H1 H1 H1 H1 H1 H1

B1 B1 B1 B1 B1 B1 B1 B1 B1

P1 P1 P1 P1 P1 P2 P2 P3 P4

S1 S1 S2 S2 S3 S4 S4 S3 S1

1 1 2 2 3 4 4 5 6

I IV I VII I I IV I II

H1 H1 H1 H1 H1 H1

B1 B1 B1 B2 B2 B2

P4 P4 P4 P5 P6 P6

S1 S2 S2 S5 S6 S6

6 7 7 8 9 9

III II III II V VI

H2

B3

P7

S7

10

VIII

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FIG. 1. Schematic representation of the seven ribotypes obtained after digestion of the Salmonella genomic DNAs with SmaI. St, molecular weight standard (Raoul, Applige`ne) revealed with radiolabeled pUC18.

separated. A unique ribotype was observed for all S. enteritidis strains. IS200 typing. Seven restriction patterns of genomic DNA for IS200 were found among the 56 S. typhimurium isolates (Table 3). The IS200 copy number ranged from six to nine. Six bands (20, 9, 4.8, 2.4, 1.9, and 1 kb) were common to the 56 S. typhimurium strains. A band of approximately 4.3 kb characterized profiles V and VI of the 10 S. typhimurium strains isolated from flocks A and B (Fig. 2) and could clearly separate them from the strains of other origins. Other bands were shared by strains isolated from different flocks. IS200 typing discriminated between strains isolated within the flocks C, E, F, H, or J. IS200 type I was found in all the strains isolated from flocks D, G, and K and in some strains isolated from flocks C, E, and J. The 14 S. enteritidis strains could not be discriminated by IS200 typing. Their IS200 type was characterized by the presence of three copies of 5.2, 4.5, and 1 kb (Fig. 3).

J. CLIN. MICROBIOL.

FIG. 3. IS200 profiles of six S. enteritidis strains. Genomic DNA digested with PstI was hybridized with the dimer of an internal fragment of IS200. Lanes 1 to 6, strains BN92R1 to BN92R3 and BN92S1 to BN92S3, respectively; lane M, molecular weight marker (Raoul, Applige`ne) hybridized with radiolabeled pUC18.

No specific hybridization was found with Southern blots of plasmid DNA. The 12 clonal S. typhimurium strains tested in the 15-week experiment presented a unique profile either by ribotyping or by IS200 typing that was identical to that of the parental strain. Data analysis. Agglomerative hierarchical cluster analysis led to the discrimination of S. typhimurium strains into three groups (Fig. 4). Correspondence analysis (data not shown) confirmed this result. In each cluster, strains presented at least 87% similarity. The major cluster was composed of 29 strains: strains isolated from flocks D, E, G, K, J, and M and strains BN91DE, BN92C8, BN92F3. The second cluster grouped all strains from flocks C, F, H, N, and P except strains BN91C6, BN92C8, and BN92F3. The third one grouped strains isolated from flocks A and B and strains BN91C6 and BN93L1. DISCUSSION

FIG. 2. Schematic representation of the seven hybridization patterns for IS200 obtained after cleavage of genomic DNA of the S. typhimurium isolates with PstI. St, molecular weight standard (Raoul, Applige`ne) revealed with radiolabeled pUC18.

Serological and biochemical typing of Salmonella isolates can be considered a first step in classification (27). Four biotypes were identified among the S. typhimurium isolates; 29 of the 56 strains belonged to the major biotype. Antibiotic resistance was of little value because of the low number of resistant strains. Plasmid profile typing, which is used for epidemiological purposes to discriminate Salmonella strains from unrelated origins (4, 12, 14), was useful for discriminating S. typhimurium strains isolated from flocks closely related or within a flock (flock C), as has already been observed (1). The strains could generally be grouped according to their place of isolation. Seven of the 10 strains isolated from flocks A and B had a unique plasmid profile that was shared only with the unrelated strain BN93P1. A unique plasmid profile and a particular antibiotic resistance pattern were observed in strains from flock J, which were discriminated by their biotypes. Strains isolated from flocks C to H, located in the same place, had plasmid profiles with a low level of diversity; this was confirmed by plasmid DNA restriction analysis. Discrimination was observed for strains BN91C6 and BN92C8 isolated from the same flock (flock C). The ribotypes of S. typhimurium strains were consistent with

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FIG. 4. Single linkage dendrogram showing the results of the cluster analysis on the basis of combined rDNA patterns and IS200 types of the S. typhimurium strains listed in Table 1.

their origins except for strains isolated from flocks C and F. Only the grouping of strains BN91C6 and BN93L1 with unrelated strains isolated from flocks A and B is surprising, but strain BN91C6 was separated from the other isolates of flock C by any method used. Like ribotyping, IS typing allowed discrimination between isolates from the same flock (flocks E and J). All strains except strain BN91A2 isolated from flocks A and B shared the same IS200 type (type V); strain BN91A2 was not separated by ribotyping and its IS type (type VI) was very close to IS type V. Strain BN91DE, isolated from the parents of ducks from flocks D and E, shared the same IS200 type (type I) with strains BN92D1 to BN92E2. This IS type was also encountered in strains BN92G1 to BN93G11, BN92K1 to BN93K6, BN92C8, and BN92J2, which were isolated in the same place. Moreover, IS types I to IV were close to each other and were found in strains isolated from flocks C to L. These results illustrate the high degree of genetic homogeneity of all of the strains isolated from flocks reared in the same area. The combination of both methods allowed the definition of 15 clonal lineages among the 56 S. typhimurium isolates. Although no difference in IS type was recorded over 15 weeks in the strain contaminating monoxenic animals, it appeared to be safer to consider strains that shared greater than 95% similarity as identical or of sufficiently close relatedness to be considered to correspond to the same clone. Clones belonging to the same cluster shared greater than 87% similarity and were thus genetically close to each other. Turkeys in the first geographical location studied carried a single clone belonging to cluster 3, which was isolated from both flocks A and B. Thorough disinfection of the premises and a general improvement of hygienic conditions are recommended. The presence of the same clone in both flocks also

points out the possible cross-contamination between flocks and the need to control the circulation of material and humans between them. Except for flocks C and F, strains isolated from one flock could be grouped within one cluster. Several independent contaminating events can be suspected in flock C, in which six of the eight isolates belonged to cluster 1 but BN91C6 and BN92C8 belonged to clusters 3 and 2, respectively. Three different clones belonging to two clusters were isolated from flock F. These flocks appear to be the most prone to external recontamination. Flocks E and J were characterized by the presence of a particular clone in each flock. Some clones appear to have spread to several flocks at the same location. One clone from cluster 1 was recovered from flocks D, K, and F. Another clone from the same cluster was recovered from flock G and was also isolated from the parent stock of flocks D and E (strain BN91DE) but was not found later in the offspring. A diffusion of strains belonging to the same cluster, and thus probably deriving from the same original strain between farmhouses of flocks D, E, G, K, and J, located in the same area, can be hypothesized. The same conclusion can be applied to strains isolated from flocks C, F, H, or A and B, respectively, with the exception of three strains in flocks C and F. The strains isolated from the related flocks M, N, and P were not clearly distinguishable from the strains isolated from flocks C to L. The level of similarity between the few strains isolated at this third geographical location is very low and is only suggestive of independent contaminating events. For IS200 typing, PstI was confirmed to be the most efficient endonuclease, providing distinct and clear patterns. In our strains, between six and nine IS200 copies were detected, in agreement with other studies (9, 11, 16). In the work of Stanley

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et al. (21), carried out on 25 S. typhimurium strains from diverse origins, three IS200 bands (4.8, 2.4, and 1.9 kb) were described as ‘‘serovar-specific’’ loci. These loci were present in all of our 56 S. typhimurium isolates. Only one S. enteritidis strain, strain BN93U1, was discriminated from the 13 other S. enteritidis strains by biotyping. It should be noted that the four quinolone-resistant strains harboring three plasmids were isolated from the fluff and meconium of newly hatched ducklings in the same flock (flock T). This probable clone was not isolated later from the reared animals, from which only quinolone-susceptible isolates harboring only one plasmid were recovered. The antibiotic resistance patterns and plasmid profile typing were thus the only discriminative methods for S. enteritidis strains. Other workers have indicated an interest in using phage typing in such epidemiological studies, but these methods are time-consuming and generally only a small number of phage types predominate in serovars such as S. enteritidis (27). Recent genetic methods did not afford separation among these strains. This confirms the high degree of genetic homogeneity previously observed for strains of that serotype (22–24) and prevents the proposal of epidemiological hypotheses. Among the 14 S. enteritidis strains, none presented IS200 copies on plasmids, in contrast to the results of Stanley et al. (24). The IS200 profile found among our isolates, with three bands of 5.2, 4.5, and 1 kb, appears to be different from those described previously: the three evolutionary lines defined by Stanley et al. (24) presented only two IS200 copies on the chromosome. Our strains, isolated from poultry in western France, belong to an original clonal lineage (CL), SECLIV, close to the clonal lineage SECLI, i.e., the CL grouping European epidemic S. enteritidis strains of PT4 (24). The IS200 type is thus a molecular marker which should complement plasmid profiling for epidemiological studies on S. enteritidis strains. No variation was detected among the 12 clones studied during a 15-week experiment, which mimics the conventional time of rearing of chickens. Only one ribotype and one IS200 type were found, both of which were identical to those detected in the parent strain. This proves the stability of the restriction patterns of chromosomal DNA for IS200. Similar results were observed with IS6110 and clones of M. tuberculosis obtained in vivo (6). Ribotyping and IS200 typing can thus be applied to epidemiological studies on Salmonella isolates, even those with a supposed clonal relatedness. For S. enteritidis strains, genotypic methods did not provide further information: phenotypic methods like plasmid profiling thus remain interesting for use in the study of such isolates in field situations. On the other hand, S. typhimurium strains can easily be separated by IS200 typing, and this method appears to be as suitable as ribotyping for epidemiological studies. Ribotyping and IS200 typing do not exactly lead to the same discrimination. This calls for the use of both typing methods in parallel when performing phylogenetic or epidemiological studies to get a very thorough discrimination. The combination with plasmid DNA analysis provides precise knowledge on the origins and degree of relatedness of the strains isolated from poultry flocks. IS200 typing appears to offer highly defined fingerprints of the S. typhimurium or S. enteritidis chromosome, is relatively simple to carry out, and can be performed more rapidly and at a lower cost than ribotyping. IS200 typing results can also be displayed in a band-matching database for strain identification, as has already been suggested by Stanley et al. (21), allowing for its use in epidemiological studies not only of regional but also of international outbreaks.

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