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Journal of Clinical Microbiology, June 2000, p. 2204-2209, Vol. 38, No. 6
Epidémiologie et Analyse des Risques,
Ecole Nationale Vétérinaire d'Alfort, F-94704
Maisons-Alfort Cedex, France
Received 16 August 1999/Returned for modification 16 November
1999/Accepted 25 March 2000
A procedure that uses an original molecular marker
(IS200-PCR) and that is based on the amplification of DNA
with outward-facing primers complementary to each end of
IS200 has been evaluated with a collection of 85 Salmonella enterica subsp. enterica serotype Typhimurium isolates. These strains were isolated from a group of 10 cows at different stages: during transportation between the farm and
the slaughterhouse, on the slaughter line, from the environment, and
from the final product (ground beef). The 85 isolates were
characterized by their antibiotic resistance patterns and were compared
by IS200-PCR and by use of four other genotypic markers.
Those markers included restriction profiles for 16S and 23S rRNA
(ribotypes) and amplification profiles obtained by different approaches: random amplified polymorphic DNA analysis, enterobacterial repetitive intergenic consensus PCR, and PCR ribotyping. The results of
the IS200-PCR were in accordance with those of other
molecular typing methods for this collection of isolates. Five
different genotypes were found, which made it possible to refine the
hypotheses on transmission obtained from phenotypic results. The
genotyping results indicated the massive contamination of the whole
group of animals and of the environment by one clonal strain originally recovered from one cow that excreted the strain. On the other hand, a
few animals and their environment appeared to be simultaneously contaminated with genetically different strains.
Salmonella spp. are some
of the most serious contaminants of food products and are the main
bacterial agent responsible for food-borne outbreaks of human
gastroenteritis in France (16). Salmonella
enterica subsp. enterica serotype Typhimurium is of particular clinical importance and is also the serotype most frequently isolated from bovine pathology material and products (5).
Bovine products are often suspected sources of human gastroenteritis when serotype Typhimurium strains are isolated. A great deal of research has been done on the contamination of meat products by Salmonella spp. However, the relationship between the
contamination of the animal before slaughter and the quality of the
final product is poorly understood. The development of molecular
markers to trace clonal Salmonella strains in order to
relate outbreaks with bovine sources is therefore important. It will
also help to trace precisely the diffusion of strains and to identify
the origins of herd contamination and therefore the origins of the
contamination of bovine products.
Numerous phenotypic and genotypic methods have thus been developed and
used in order to subtype Salmonella serotypes, particularly serotype Typhimurium isolates. Phage typing is of great usefulness for
the description of important pandemic clones, for instance, S. enterica subsp. enterica serotype Typhimurium DT104
(2, 36). Plasmid profiling has proved useful in various
epidemiological studies (24, 41). Chromosomal
characterization by ribotyping, restriction fragment length
polymorphism (RFLP) analysis for IS200, or pulsed-field gel
electrophoresis has been evaluated for the more precise subtyping of
Salmonella strains (3, 8, 14, 24). The use of PCR
with different complementary approaches has been considered recently
for Salmonella isolates, either a random approach named
random amplified polymorphic DNA (RAPD) analysis (or arbitrarily primed
PCR) (7, 13, 18, 19, 25) or methods based on repetitive
elements present in several copies on the chromosome, for example, PCR
with enterobacterial repetitive intergenic consensus (ERIC) sequences
(ERIC-PCR) (7, 19, 25) and PCR ribotyping (21, 22,
27).
A procedure for the amplification of DNA fragments with outward-facing
primers complementary to each end of IS200
(IS200-PCR) has been designed and evaluated in our
laboratory. We hypothesized that the number of copies of
IS200 as well as the IS200 insertion positions
would be strain specific and that these copies would be spaced close
enough to allow amplification of polymorphic inter-IS200 element sequences. Thus, these variations would allow different sizes
and numbers of DNA fragments to be amplified, yielding unique banding
patterns for different Salmonella strains.
IS200 is a short insertion sequence (IS) of about 708 bp
that was first described in Salmonella. It is related to
other ISs found in several other genera such as Yersinia
(IS1541) (30) and Clostridium
(IS1469) (6). IS200 has been
extensively used for the typing of Salmonella isolates of
numerous serotypes by RFLP analysis (8, 14, 24), an approach
that could be called IS200 typing (9). Similarly,
molecular typing by RFLP analysis for IS1541 of isolates of
Yersinia pseudotuberculosis has been described
(29).
The aims of the present investigation were first to evaluate
IS200-PCR for the tracing of bovine Salmonella
isolates and second to investigate more precisely the relationship
between contamination of cows and the resulting contamination of the
carcasses and ground meat produced from these animals. Eighty-five
strains of S. enterica subsp. enterica serotype
Typhimurium isolated from animals and from their environment were
characterized by phenotypic methods as well as by chromosomal
fingerprinting. IS200-PCR was compared with PCR with other
molecular markers. Ribotyping was considered the reference method in
our study.
Bacterial strains.
The reference strains used in our
experiments are presented in Table 1. F98
was kindly provided by P. A. Barrow (Houghton Laboratory, Institute for
Animal Health, Agriculture and Food Research Council, Huntingdon,
United Kingdom), and strains BN8301, BN8501, BN91A1, and BN91C1 were
kindly provided by E. Chaslus-Dancla (Institut National de la Recherche
Agronomique, Tours, France). Strains IP5210, IP5858, and IP6062 are
serotype Typhimurium isolates from the reference collection of the
Institut Pasteur (Paris, France).
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Evaluation of IS200-PCR and Comparison with Other
Molecular Markers To Trace Salmonella enterica subsp.
enterica Serotype Typhimurium Bovine Isolates from Farm
to Meat
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Source and PCR types of the S. enterica subsp.
enterica serotype Typhimurium strains studied
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Resistance to antibiotics. The strains were screened for their resistance to 16 antibiotics (Sanofi-Diagnostics Pasteur, Marnes-la-Coquette, France) by the standardized disk diffusion technique (4): amoxicillin (25 µg), florfenicol (30 µg), apramycin (10 IU), cefalexin (30 µg), ceftiofur (30 µg), cefquinome (30 µg), colistin (50 µg), enrofloxacin (5 µg), flumequine (30 µg), gentamicin (10 IU), neomycin (30 IU), oxolinic acid (10 µg), spectinomycin (100 µg), streptomycin (10 IU), tetracycline (30 IU), and trimethoprim-sulfamethoxazole (1.25/23.75 µg).
Restriction profiles for rDNA. To obtain restriction profiles, total DNA was extracted from the bacteria after an 18-h culture at 37°C either by a modified version of the salting-out technique described by Miller et al. (26) or as described by Wilson (40). Two to 5 µg of DNA was digested with 10 U of a restriction endonuclease (Boehringer Mannheim, Mannheim, Germany). Four different endonucleases were chosen for ribotyping after preliminary experiments: HindIII, PstI, PvuII, and SmaI. Genomic restriction digests were electrophoresed on 0.8% horizontal agarose gels and were visualized with shortwave UV light. The Raoul marker (Appligène, Illkirch, France) was used as a molecular weight standard. Afterward, the digestion products were vacuum blotted (Pharmacia Biotech, Uppsala, Sweden) onto Hybond-N nylon membranes (Amersham, Little Chalfont, United Kingdom). Blots of digested electrophoresed DNA were hybridized with the p14b1 plasmid containing a Bacillus subtilis ribosomal DNA (rDNA) insert (17). Probes were digoxigenin labeled by using the DIG high prime labeling and detection starter kit I (Boehringer Mannheim). Hybridization was conducted at 65°C in a hybridization oven (Techne, Cambridge, United Kingdom). The filters were stringently washed. Homologous bands were visualized after revelation with nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate (Boehringer Mannheim).
PCR methods. (i) DNA extraction. For amplification profiles, DNA was extracted by a boiling method as follows: 1.5 ml of an overnight broth culture was centrifuged at 15,000 × g for 10 min. The pellet was resuspended twice in 100 µl of sterile ultrapure water and was then boiled for 10 min. After a final centrifugation at the speed mentioned above, the supernatant was gently recovered.
(ii) Primer design. For IS200-PCR, we used the PileUp program of the GCG package. A multiple alignment was performed with a set of Salmonella IS200 sequences found in the GenBank database (accession numbers X56834, Y09564, U44749, Z54217, Y09991, Y09990, Y09989, X91136, L25848, M57304, M57306, X03452, and X03451). Primers corresponding to the most conserved regions were designed to be outward facing and complementary to each end of the consensus sequence of IS200. Primers and primer sequences were as follows: IS1R, 5'-AGGCGCATCTGAAAAACCTCGG-3' (nucleotides 667 to 688), and IS2, 5'-CGGAACCCCCAGCCTAGCTGGG-3' (nucleotides 35 to 14).
For RAPD analysis, preliminary assays were conducted with 60 decanucleotides from F, G, and H kits (Operon Technologies, Alameda, Calif.) with the eight reference strains. Five primers were chosen among the 60 decanucleotides for subsequent studies because of the clear and distinct banding patterns obtained: OPF08 (5'-GGGATATCGG-3'), OPF13 (5'-GGCTGCAGAA-3'), OPG04 (5'-AGCGTGTCTG-3'), OPG10 (5'-AGGGCCGTCT-3'), and OPH04 (5'-GGAAGTCGCC-3'). The primers used for ERIC-PCR are based on ERIC sequences and have been described by Versalovic et al. (38). When performing PCR-ribotyping, we used the primers described by Kostman et al. (20) in order to amplify 16S-23S intergenic sequences.(iii) PCR conditions. PCR conditions were as follows. Each set of reactions included a control tube without template DNA. For each method used, the amplification conditions (deoxynucleoside triphosphate concentration, annealing temperature, duration of extension, DNA concentration) have been varied in order to obtain reproducible banding patterns. For all methods used in this study, we chose to add 1 U of Taq DNA polymerase (Bioprobe Systems, Montreuil, France) in a 25-µl reaction volume and to work with a final deoxynucleotide triphosphate concentration of 0.2 mM (Bioprobe Systems) in the recommended reaction buffer containing 20 mM Tris HCl, 16 mM (NH4)2SO4, 2.5 mM MgCl2, and 0.2 µg of bovine serum albumin per ml. In all cases, 10 ng of genomic DNA was added to each tube. All amplifications were performed in a PHC-3 thermal cycler (Techne Instruments, Cambridge, United Kingdom).
For the IS200-PCR, 20 pmol of each outward-facing primer, IS1R and IS2, was added to the 25-µl reaction volume. The optimal amplification cycles for IS200-PCR were as follows: 94°C for 3 min, 55°C for 1 min, and 72°C for 4 min for the first cycle and then 94°C for 1 min, 55°C for 1 min, and 72°C for 4 min for the other 45 cycles. The final cycle included 1 min of denaturation at 94°C, 1 min of annealing at 55°C, and a final extension at 72°C for 20 min. For the RAPD technique, 15 pmol of one primer was added to the reaction volume. Amplification cycles for RAPD analysis were as follows: 94°C for 3 min, 35°C for 30 s, and 72°C for 1 min 30 s for the first cycle and 94°C for 40 s, 35°C for 30 s, and 72°C for 1 min 30 s for the other 35 cycles. The final extension at 72°C lasted 10 min. For performance of the ERIC-PCR, 12.5 pmol each of ERIC1R and ERIC2 primers was added to the reaction volume. Amplifications cycles for ERIC-PCR were as follows: 94°C for 3 min, 51°C for 30 s, and 72°C for 4 min for the first cycle and then 94°C for 1 min, 51°C for 1 min, and 72°C for 4 min for the other 35 cycles. The final cycle included 1 min of denaturation at 94°C, 1 min of annealing at 51°C, and a final extension at 72°C for 20 min. For PCR ribotyping, 25 pmol each of primers SP1 and SP2 was added in a 25-µl reaction volume. Amplification cycles for PCR-ribotyping were as follows: 94°C for 3 min, 55°C for 1 min, and 72°C for 1 min for the first cycle and then 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min for the other 29 cycles. The final cycle included 1 min of denaturation at 94°C, 1 min of hybridization at 55°C, and a final extension at 72°C for 4 min. Amplification products were separated by horizontal electrophoresis through 2% agarose gels. Marker VIII (Boehringer Mannheim) was used as a molecular weight standard.Reproducibility. The reproducibilities of the fingerprints were assessed with successive runs with the same samples and also with different DNA samples extracted from a single isolate.
Data analysis. The rDNA patterns and PCR types were analyzed as described previously (24, 25), except that agglomerative hierarchical cluster analysis was performed by using the commercial software program SAS (1996; SAS Institute Inc., Cary, N.C.).
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RESULTS |
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Introduction
Materials and Methods
Results
Discussion
References
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Resistance to antibiotics. All 85 strains isolated in the field study harbored the same antibiotype, being resistant only to amoxicillin and tetracycline. They were sensitive to all the other 14 antibiotics tested.
Comparison of methods of S. enterica subsp. enterica serotype Typhimurium typing. A number of different fingerprints were obtained by each method used (Table 1).
Reference strains of different geographical and animal origins could be separated by three of the four PCR methods evaluated. Two patterns were obtained by IS200-PCR, with strains IP5858 and IP6062 being differentiated from the others. RAPD analysis proved to be the most discriminative and allowed assignment of a distinct pattern to each strain, after combination of the results obtained with the five primers retained after preliminary assays. Primer OPG04 was the most discriminative, allowing the observation of five distinct profiles, while OPG10 provided three patterns. Only two patterns were obtained with each primer OPF08, OPF13, or OPH04, but a different pattern was discriminated by each primer. Four different PCR ribotypes were identified. ERIC-PCR provided only one profile for the eight reference strains used in this study. In our study with field isolates, IS200 PCR resulted in three different fingerprints. They exhibited six to eight bands whose molecular sizes were between 240 bp and 1 kb (see Fig. 1, which presents the fingerprints observed among the 85 isolates studied by IS200-PCR). LV3 had a unique pattern (profile C), while LV26, LV33, and LV55 shared profile D. All other isolates studied exhibited profile B.
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DISCUSSION |
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Materials and Methods
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Discussion
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The use of PCR-based methods for the typing of bacterial isolates is promising because they are simple to carry out and provide a rapid answer which is easy to interpret. They are generally considered to be able to provide a high degree of discrimination at a low cost (31). By contrast, ribotyping, which is generally considered a reference technique, remains a time-consuming and expensive method.
PCR amplification of segments located between distinct repetitive DNA elements allows the development of rapid methods useful for the subtyping of bacteria. For instance, ERIC-PCR relies on PCR amplification of fragments located between ERIC sequences with outward-facing primers complementary to each end of the ERIC sequences (38). A similar approach named "inter-IS256 spacer analysis" has been developed for the typing of Staphylococcus isolates, which led to results similar to those obtained by other molecular typing methods (11). Several related methods have been evaluated for the subtyping of Mycobacterium isolates. Ross and Dwyer (37) used the terminal regions of IS6110 to divergently amplify DNA segments between copies of this insertion sequence. Similarly, Mycobacterium avium isolates have been typed by a rapid technique based on PCR amplification of genomic sequences located between two related insertion sequences: IS1245 and IS1311 (33). This technique has been evaluated by Yoder et al. (43) for the comparison of levels of relatedness of M. avium isolates found in patients and foods. Those investigators concluded that this PCR typing method was a rapid, inexpensive method for the typing of M. avium, possibly replacing pulsed-field gel electrophoresis. While the latter rapid method used two primers, Otal et al. (32) proposed the use of only one primer for the amplification of segments located between IS6110 copies and compared this technique, called "IS6110-PCR," to "IS6110-inverse-PCR," in which amplification followed digestion and ligation. They compared IS6110-inverse-PCR and IS6110-PCR to IS6110-RFLP analysis and found concordant results: they recommended IS6110-PCR as a screening method for the quick differentiation between M. tuberculosis strains. Plikaytis et al. (34) developed a method called "ampliprinting," based on PCR amplification of spacers between IS6110 elements and copies of a major polymorphic tandem repeat sequence (related to repetitive extragenic palindromic sequences of Escherichia coli) in Mycobacterium tuberculosis. The double-repetitive-element PCR based on amplification of spaces between IS6110 copies and the polymorphic GC-rich repetitive sequence, as well as RFLP methods, has been shown to be able to differentiate M. tuberculosis isolates (15).
A method for the amplification of DNA fragments with outward-facing primers complementary to each end of IS200 (IS200-PCR) has been designed and evaluated in our laboratory. In our field study, the results of IS200-PCR corroborated the results obtained with the other markers assayed and thus reinforced the proposed epidemiological hypotheses. It can be noted that in our work the number of bands amplified by IS200-PCR (six to eight) is compatible with the number of IS200 copies generally found on the chromosomes of S. enterica subsp. enterica serotype Typhimurium isolates (24). IS200-PCR appears to be a novel and rapid method which discriminates among bovine field isolates in accordance with the discriminatory abilities of other markers. It must be underlined that the results obtained by PCR typing methods were consistent with those of ribotyping, with the latter method being considered the reference method. IS200-PCR provided a rapid answer, at a low cost, and patterns that were easy to read, without a preliminary search for oligonucleotides, in contrast to RAPD assays (22, 25). Thus, we suggest that IS200-PCR could provide a rapid and simple means of typing Salmonella isolates for epidemiological studies.
Yet, IS200-PCR will be applicable only to epidemiological studies with Salmonella strains which harbor copies of the IS200 element on their chromosomes. For instance, it will not be applicable to S. enterica subsp. enterica serotype Hadar isolates because of the lack of such elements in isolates of this serotype (39). However, a comparative study was carried out with five S. enterica subsp. enterica serotype Dublin isolates from our collection. Three specific fingerprints were generated by IS200-PCR, which allowed differentiation among serotype Dublin isolates. The fingerprints of serotype Dublin and Typhimurium strains were easily distinguished, although they shared three bands of similar molecular weights (data not shown).
The results of typing of 85 bovine S. enterica subsp. enterica serotype Typhimurium isolates led us to propose epidemiological hypotheses concerning the contamination of animals during transport and before slaughter as well as the contamination of the final product.
Initially, phenotypic characterization was achieved by serotyping and determination of resistance to antibiotics. Only one antibiogram could be detected, with resistance to amoxicillin and tetracycline. Resistance to these antibiotics is widely encountered in bovine serotype Typhimurium isolates. No clear conclusion could be drawn on the basis of these homogenous phenotypic characteristics.
The molecular characterization refined the results and discriminated five isolates according to their PCR types. This grouping was thereafter confirmed by classical ribotyping (considered a reference method). On the basis of these results, it appears that the major clone had initially been recovered on the farm and was harbored by animal 5. Thereafter, 79 other isolates were isolated from the 10 animals and from the environment. This led to the hypothesis of contamination, probably at a high level, of the whole group with a single major clone.
Moreover, four minor clones were recovered at different steps and/or were excreted by different animals (Tables 1 and 2). Knowledge of their precise sources with the help of the analysis of the dendrogram (which showed clonal ties between isolates) allows us to formulate some hypotheses. Isolate LV3 was isolated in the feces of animal 3. It was very different from the major clone (the genetic distance between them is large). This particular clone has been isolated only once. It is possible that it was excreted at a low concentration, preventing widespread diffusion. The LV6 strain is genetically closely related to the major clone, although it was isolated in the environment of the abattoir before the arrival of the animals. It may have been brought to the abattoir by animals of the same geographical origin. LV26 was isolated on the ground of the cubicle with animal 6, LV33 was isolated from the environment of animal 3, and LV55 was isolated in the stunning area after animal passage. LV26, LV33, and LV55 isolates share 85% similarity. As they were recovered in the environment of the abattoir, all three isolates could be resident strains of the abattoir.
Finally, numerous strains have been isolated during the experiment: this suggests that the excreting animal contaminated its environment as well as the other animals to a large extent. It may be hypothesized that this animal excreted a large amount of salmonellae: this could be related to transport-related stress. The transportation period is in fact considered to be important for the amplification of Salmonella excretion and dissemination (10, 42). As the same clone was isolated starting from the farm up to the final meat product, the risk of meat contamination is underlined, and therefore the risk of human food-borne disease not only from a Salmonella carrier animal but also from animals that were transported with this carrier is increased. This potential excretion of Salmonella by cattle must be considered, for instance, in order to reorganize slaughtering to limit the risk of contamination. It could be suggested that potential Salmonella excretors be slaughtered at the end of the day or, better, be removed from the production of ground meat.
A difficulty of epidemiological typing is the choice of a "gold standard" that may take into account the evolution of the strains. For instance, although RFLP analysis with IS6110 has been shown to be stable in vitro and in vivo, Alito et al. (1) recently described nine M. tuberculosis isolates from related subjects with slightly different RFLP patterns. Thus, they concluded that the IS6110 RFLP in particular multidrug-resistant M. tuberculosis strains may evolve too fast for reliable use when studying outbreaks.
It could be interesting to locate on the genome the regions in which the epidemiological markers used have detectable differences. In fact, genetic rearrangements that are responsible for the evolution of chromosomal fingerprints can occur. Such rearrangements between rrn operons that result in modifications of the ribotype have been demonstrated in Salmonella serovar Typhi (28) as well as in other serotypes including Typhimurium (23). In fact, chromosomal rearrangements have also been shown to occur during the emergence of an outbreak, affecting molecular typing of Salmonella serovar Typhi strains (12). A better understanding of the consequences of such modifications must be achieved in order to evaluate more objectively and more precisely the genetic relationships between isolates for epidemiological purposes.
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ACKNOWLEDGMENTS |
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The Ministry of Agriculture with the financial support of ARCADIE SA funded the work.
We thank D. Marc, whose help in conceiving primers IS1 and IS2R was accurate, and R. Aufrere, who kindly provided plasmid p14b1. O. Cerf and P. Pardon are gratefully acknowledged for advice. M. Cance (APAVE) and A. Laval (ENV Nantes) are gratefully acknowledged for collaboration. We also thank Andrew Ponter (ENV Alfort) and Mark Tepfer (INRA Versailles) for critical reading of the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: Département des Productions Animales et des Sciences des Aliments, Epidémiologie et Analyse des Risques, Ecole Nationale Vétérinaire d'Alfort, 7 av. du Général de Gaulle, 94704 Maisons-Alfort Cedex, France. Phone: (33) (0)1 43 96 71 23. Fax: (33) (0)1 43 96 70 24. E-mail: millemann{at}vet-alfort.fr.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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