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Journal of Clinical Microbiology, September 2000, p. 3484-3488, Vol. 38, No. 9
Field Disease Investigation Unit, College of
Veterinary Medicine,1 Department of
Microbiology, College of Sciences,2 and
Department of Veterinary Microbiology and
Pathology,3 College of Veterinary Medicine,
Washington State University, Pullman, Washington 99164
Received 2 March 2000/Returned for modification 24 April
2000/Accepted 21 June 2000
A DNA sequence was identified in isolates of Salmonella
enterica serotype Typhimurium definitive type 104 (DT104). The
PCR amplification of an internal segment of this sequence identified DT104 and the closely related U302 phage type among 146 isolates of
S. enterica serotype Typhimurium tested, thus providing a
tool for rapid identification of DT104 and related isolates.
An epidemic strain of
Salmonella enterica serotype Typhimurium definitive type 104 (DT104) rose to prominence when this multidrug-resistant (R-type
ACSSuT) pathogen was identified as a major cause of salmonellosis in
people and farm animals in Britain (12) and in people in the
United States (5). The epidemic strain of DT104 was defined by British researchers based upon Salmonella serovar
Typhimurium phage type 104, the R-type ACSSuT, and a plasmid profile
consisting of a single ~60-MDa plasmid (12).
Antimicrobial susceptibility tests to determine R-type (2)
and plasmid profile analyses (10) are widely available in U.S. laboratories, but identification of S. enterica
serotype Typhimurium phage types requires the maintenance of a phage
library and specially trained personnel. Thus, phage typing will likely always be limited to a few centralized laboratories. In addition, some
isolates cannot be assigned a phage type with the available phage
library. These difficulties hinder the progress of epidemiological investigations. We describe in this note the identification of a DNA
sequence that is unique to the DT104 and U302 phage types among
S. enterica serotype Typhimurium isolates, and the
development of a PCR assay to identify isolates containing this
sequence. This PCR assay can be used to rapidly screen suspect samples
or isolates for further testing and identification.
The PCR amplification of the 16S-to-23S spacer region of bacterial rRNA
genes has been used to detect polymorphisms in bacterial species and to
identify strains of S. enterica serotype Typhimurium associated with disease outbreaks (8, 9). Using this
technique on bovine isolates of S. enterica serotype
Typhimurium, the DT104 isolates demonstrated a unique band compared to
other phage types (Fig. 1). This unique
band was purified from the agarose gel and sequenced by the Laboratory
for Bioanalysis at Washington State University. The sequence data are
in boldface in Fig. 2.
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Identification of DT104 and U302 Phage Types among
Salmonella enterica Serotype Typhimurium Isolates by
PCR
ABSTRACT
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FIG. 1.
PCR amplification products of isolates of S. enterica serotype Typhimurium obtained with primers to the
conserved regions of the 16S and 23S ribosomal genes as described by
Kostman et al. (8) and analyzed by 2% agarose gel
electrophoresis and staining with ethidium bromide. Lanes: 1 to 3, DT104; 4, 123-bp DNA ladder (Life Technologies, Gaithersburg, Md.); 5, phage type 1; 6, phage type 193. The ~550-bp polymorphism of the
DT104 isolates is indicated by the arrow.
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FIG. 2.
The 1,767-bp sequence identified in S. enterica serotype Typhimurium DT104. Boldface bases indicate the
sequence identified as a polymorphism in Fig. 1 that was the result of
the primers to the rRNA gene sequences annealing to nonribosomal
sequences (identified by dotted arrows
[·········>]). The positions of
primers DT104F and DT104R are indicated by solid arrows
( 
). The positions of additional primers designed to
extend the sequence are indicated by dashed arrows
(--->).
The original sequence was extended in both the 5' and 3' directions by using sequence-specific primers (Fig. 2) and a kit designed to facilitate genomic walking (The Vectorette System; Genosys, The Woodlands, Tex.). A search of the nucleotide sequence databases did not produce any matches to known ribosomal sequences, demonstrating that the amplified polymorphism identified in Fig. 1 was the result of the annealing of the primers to nonribosomal sequences. The possible locations of the annealing sites are indicated on the sequence in Fig. 2.
To determine the association of this sequence with S. enterica serotype Typhimurium DT104, primers were designed for PCR amplification of an internal segment of the sequence. Figure 2 illustrates the locations of the primers DT104F (5'-GTCAGCAGTGTATGGAGCGA-3') and DT104R (5'-AGTAGCGCCAGGACTCGTTA-3'), which were designed to amplify a 162-bp segment. In addition, primers INVA-1 (5'-ACAGTGCTCGTTTACGACCTGAAT-3') and INVA-2 (5'-AGACGACTGGTACTGATCGATAAT-3'), which amplify a 243-bp segment of the Salmonella invA gene (4, 11), were included in the PCR assay as a positive control for sample preparation and the amplification reaction.
To evaluate the multiplex PCR, 239 Salmonella isolates representing a wide range of serotypes and sources were selected from a bank of Salmonella isolates maintained by the Field Disease Investigation Unit (FDIU), College of Veterinary Medicine, Washington State University, Pullman, Wash. These isolates were associated with clinical cases of salmonellosis and were collected by the FDIU or submitted to the Washington Animal Diseases Diagnostic Laboratory between 1986 and 1997. All isolates were serogrouped and tested for antimicrobial susceptibility (2) by the FDIU. Serotypes were determined by the National Veterinary Services Laboratory (NVSL), U.S. Department of Agriculture, Ames, Iowa. Phage typing was performed with 57 of the 146 isolates of S. enterica serotype Typhimurium by the National Laboratory for Enteric Pathogens, Health Canada, Ottawa, Canada (6), or the NVSL. Plasmid profiles (10) for 21 isolates of S. typhimurium phage type 104 were determined by the FDIU to confirm these isolates were of the epidemic strain.
Cell lysates for each isolate were prepared in duplicate by suspending
a single bacterial colony in 300 µl of sterile distilled water in a
microcentrifuge tube and boiling for 20 min. Cell lysates were stored
at 
20°C until amplified. Aliquots (5 µl) of cell lysates were
each amplified in a 25-µl reaction mixture with 1 µM (each) primers
INVA-1, INVA-2, DT104F, and DT104R; 200 µM (each) deoxynucleoside
triphosphates (dNTP), 2 mM MgCl2, 20 mM Tris-HCl (pH 8.4),
50 mM KCl, and 1.25 U of Taq DNA polymerase (Life
Technologies, Gaithersburg, Md.). Amplification was performed in 0.2-ml
microreaction tubes in a RapidCycler (Idaho Technology, Idaho Falls,
Idaho) as follows: denaturation at 96°C for 1 min; 30 cycles of
96°C for 30 s, 60°C for 30 s, and 72°C for 35 s;
and 1 final extension cycle at 72°C for 30 s. The PCR products
were visualized on ethidium bromide-stained agarose gels (Fig.
3).
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Table 1 illustrates the variety of
serogroups, serotypes, and sample sources tested by the multiplex PCR
assay. In addition to the S. typhimurium DT104 isolates, 15 isolates of other serotypes produced the 162-bp product after PCR
amplification, including S. enterica serotype Brandenburg (1 of 1), S. enterica serotype Lille (3 of 3), S. enterica serotype Muenchen (1 of 2), S. enterica serotype Dublin (9 of 9), and S. enterica serotype
Enteritidis (1 of 3). The amplification product from one representative
isolate of each positive serotype was purified and sequenced. The
sequence data in Fig. 4 demonstrate the
PCR assay amplifies a similar 162-bp segment of DNA in these diverse
serotypes.
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Table 2 illustrates that among the 54 isolates of S. enterica serotype Typhimurium with phage
typing data, all 21 DT104 isolates tested by this multiplex PCR
produced the 162-bp amplification product. In addition, three isolates
with phage type U302 produced the 162-bp amplification product, and
U302 is considered an offspring of the DT104 strain (R. D. Khakhria, personal communication). None of the 15 other defined phage
types of S. enterica serotype Typhimurium tested produced
the 162-bp product following PCR. Three isolates of multidrug-resistant
S. enterica serotype Typhimurium submitted for phage typing
could not be assigned a defined phage type, and these isolates also did
not produce the 162-bp product following PCR.
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A sequence identity search (1) produced a closely matched
alignment (98% identity) between bases 1645 and 1716 of the newly identified sequence in S. enterica serotype Typhimurium
DT104 and 71 of 72 bases of an unfinished fragment of S. enterica serotype Typhi (microbial genome BLAST database accession
no. Sanger_601). An additional alignment (80% identity) was identified
for 382 bp of the newly identified sequence with a cDNA clone from a
mouse blastocyst that is described as being similar to replication
protein 14 from bacteriophage 
80 (GenBank accession no. AA574821).
Recent studies have described PCR assays to detect S. enterica serotype Typhimurium DT104 based upon amplification of
regions of antibiotic resistance genes (3, 7). Our data
indicate that the sequence we have identified in DT104 is not
associated with the R-type ACSSuT. Fifteen isolates among other
serotypes yield an amplification product similar to DT104, but these
isolates were predominantly sensitive to one or more of the antibiotics that distinguish multidrug-resistant DT104. There was no common resistance pattern among these non-Typhimurium-positive isolates, and 5 of the 15 isolates were sensitive to all five antibiotics. Also, five
isolates of S. enterica serotype Typhimurium
one phage type
10, one phage type 771, and three isolates that could not be phage
typedall have the R-type ACSSuT and do not yield an amplification
product in our PCR assay (Table 2).
In conclusion, we have identified a 1,767-bp sequence in S. enterica serotype Typhimurium DT104 and developed a PCR assay to amplify an internal segment of this sequence. This multiplex PCR assay, in combination with serotype data, identifies the DT104 and U302 phage types. Furthermore, the similarity of the 162-bp amplification products among different serotypes of Salmonella and the alignment of a portion of the extended sequence with an unfinished fragment of S. enterica serotype Typhi suggest the possibility of the horizontal transfer of DNA.
Nucleotide sequence accession number. The sequence of DT104 has been submitted to GenBank under accession no. AF275268.
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ACKNOWLEDGMENTS |
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This work was funded by a grant from the American Veterinary Medical Foundation and the USDA Fund for Rural America.
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FOOTNOTES |
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* Corresponding author. Mailing address: Field Disease Investigation Unit, College of Veterinary Medicine, Washington State University, Pullman, WA 99164. Phone: (509) 335-0808. Fax: (509) 335-0880. E-mail: lcp{at}vetmed.wsu.edu.
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Journal of Clinical Microbiology, September 2000, p. 3484-3488, Vol. 38, No. 9
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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