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Dispatch
Role of Electronic Data Exchange
in an International Outbreak Caused by Salmonella enterica Serotype
Typhimurium DT204b
Elizabeth A. Lindsay,*Andrew J. Lawson,* Rachel A. Walker,* Linda
R. Ward,* Henry R. Smith,* Fiona W. Scott,* Sarah J. O'Brien,† Ian S.T.
Fisher,† Paul D. Crook,†Deborah Wilson,‡ Derek J Brown,§ Hjordis Hardardottir,¶
Wim J.B. Wannet,** Helmut Tschäpe,†† and E. John Threlfall*
*Public Health Laboratory Service Laboratory of Enteric Pathogens,
London, United Kingdom; †Public Health Laboratory Service Communicable
Diseases Surveillance Centre, London, United Kingdom; ‡County Durham and
Darlington Health Authority, Durham, United Kingdom; §North Glasgow University
Hospitals National Health Service Trust, Glasgow, Scotland; ¶Landspitali
University Hospital, Reykjavik, Iceland; **National Institute of Public
Health and the Environment, Bilthoven, the Netherlands; and ††Robert-Koch
Institut, Harz, Germany
From July through
September 2000, patients in five European countries were infected
with a multidrug-resistant strain of Salmonella Typhimurium
DT204b. Epidemiologic investigations were facilitated by the transmission
of electronic images (Tagged Image Files) of pulsed-field gel electrophoresis
profiles. This investigation highlights the importance of standardized
protocols for molecular typing in international outbreaks of foodborne
disease.
The Study
From July through September 2000, patients in five European countries
(England, Scotland, Germany, the Netherlands, and Iceland) were infected
with a strain of Salmonella enterica serotype Typhimurium definitive
phage type (DT) 204b; the strain was resistant to ampicillin (A), chloramphenicol
(C), gentamicin (G), kanamycin (K), streptomycin (S), sulphonamides (Su),
tetracyclines (T), trimethoprim (Tm), and nalidixic acid (Nx). The strain
also had decreased susceptibility to ciprofloxacin (CpL), with
an MIC by E-test of 0.38 mg/L (1,2). Over 350 laboratory-confirmed
cases were recognized. Epidemiologic investigations implicated shredded
lettuce as the vehicle of infection (1).
Isolates from patients in Iceland, the Netherlands, and Scotland were
referred to the England and Wales Public Health Laboratory Service (PHLS)
Laboratory of Enteric Pathogens for phage typing and antibiogram analysis.
These isolates were compared with those from a concurrent outbreak of
multiresistant S. Typhimurium DT204b in northeastern England and
from patients returning to England and Wales after visiting other European
countries. A panel of seven isolates from outbreaks in England, Scotland,
Iceland, and the Netherlands and from patients returning to the United
Kingdom after visiting Greece, Germany, and the Netherlandswere further
characterized by a variety of molecular techniques, including plasmid
profile typing, pulsed-field gel electrophoresis (PFGE), fluorescent amplified
fragment-length polymorphism fingerprinting (FAFLP), and integron typing;
specific resistance genes were characterized by polymerase chain reaction
(PCR) and the mutation conferring decreased susceptibility to ciprofloxacin
was identified by a LightCycler (Roche Diagnostics Ltd., Lewes, U.K.)
gyrAmutation assay (GAMA) (3). Isolates from Germany
and Scotland were typed independently with the same phenotypic methods
as those used in the Laboratory of Enteric Pathogens and also by plasmid
profile typing and PFGE. To facilitate epidemiologic investigations, PFGE
Tagged Image Files (TIFs) of banding patterns of isolates from England,
Scotland, and Germany were exchanged electronically.
Conjugation experiments were performed at both 28°C and 37°C by using
a rifampicin-resistant strain of Escherichia coli (strain 20R764)
as the recipient. Resultant plasmids were classified by incompatibility.
DNA was extracted from transconjugants by using a DNeasy tissue kit (Qiagen
Ltd., Crawley, U.K.). For plasmid profile analysis, plasmids were resolved
by electrophoresis at 110 V for 3 hours in 0.8% wt/vol agarose. Oligonucleotide
primers synthesized by MWG-Biotech UK Ltd. (Milton Keynes, U.K.) were
used to detect the antibiotic resistance genes aadA2, blaCARB-2,
blaTEM, sul1, tetA (class A), tetA
(class G), tetA (class B), and integrons in both wild-type strains
and E. coli K12 transconjugants. The nucleotide sequence of these
primers and the corresponding temperature profiles for amplification have
been described (4). GAMA, designed to detect three different
gyrA mutations, was performed in a LightCycler instrument under
previously described reaction components and conditions (3).
For PFGE, agarose plugs were prepared by the method of Powell et al. (5)
with 2% chromosomal grade agarose (Bio-Rad Laboratories, Hemel Hempstead,
U.K.) replacing the 2% Type VII LGT agarose (Sigma Chemical Co., Poole,
U.K.). Samples were run through a 1% pulsed-field certified agarose gel
(Bio-Rad) at 180 V for 44 hours, with pulse times ramped from 6 to 72
seconds. For FAFLP, the selective primer combinations Eco+0 Mse+T,
Eco+0 Mse+TA, and Eco+0 Mse+CG were used,
and gel separation and fragment analyses were performed, all as described
by Scott et al. (6).
All seven isolates had phage typing reactions corresponding to S.
Typhimurium DT204b. This rare phage type was reported in only 40 cases
in England and Wales in the 5-year period 1996–2000 and had never been
identified in Iceland before this epidemic.
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Click to view
enlarged image
Figure.
Pulsed-field gel electrophoresis profiles of XbaI-digested
genomic DNA from isolates of Salmonella enterica serotype
Typhimurium DT204b.
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With the exception of resistance to nalidixic acid and decreased susceptibility
to ciprofloxacin, the complete resistance spectrum (ACGKSSuTTm) was transferable
to E. coli K12 rifr as an intact linkage group. Plasmid
profile analysis demonstrated the presence of five plasmids of 120, 65,
4.0, 3.0, and 2.0 MDa. Most transconjugants with the resistance pattern
ACGKSSuTTm had a single incompatibility group H2 plasmid of
120 MDa. A few transconjugants had both the 120-MDa plasmid and an additional
plasmid of 65 MDa. When PCR amplification was performed on DNA from transconjugants
harboring either the single 120-MDa plasmid or both the 120-MDa and 65-MDa
plasmids, positive results for aadA2, blaTEM,
sul1, and tetA (class A) were obtained; blacarb-2,
tetA (class G), and tetA (class B) were negative. When studied
by integron PCR, one discrete 1.6-kb band was generated in transconjugants
when either the 120-MDa plasmid or both the 120-MDa and the 65-MDa plasmids
were present. In addition, one very faint amplicon of approximately 4
kb was consistently produced. Whether both the 120-Mda plasmid and the
65 Mda-plasmid have tetracycline resistance genes or whether tetracycline
resistance is encoded by only the 120-MDa plasmid is unclear. When studied
by GAMA, the gyrA mutation was that of aspartate to glycine (GAC-GGC)
at codon 87.
PFGE profiles of all seven isolates were indistinguishable (Figure).
To facilitate epidemiologic investigations, PFGE and plasmid profile TIFs
of the banding patterns of these isolates and those of multiresistant
S. Typhimurium DT204b from Scotland and Germany were exchanged
electronically. In all cases, the resultant PFGE and plasmid profiles
were indistinguishable. Finally, when studied by FAFLP, the seven isolates
were identical.
Conclusions
These results confirm that the strains of S. Typhimurium DT204b
of R-type ACGKSSuTTmNxCpL responsible for outbreaks of infection
in five European countries in the summer of 2000 were indistinguishable
by all phenotypic and molecular criteria used for their characterization.
A key aspect of this investigation was the rapid exchange of molecular
fingerprints between laboratories already using standardized phage typing
and antimicrobial susceptibility testing. In the United States, the exchange
of molecular data has been addressed by the establishment of PulseNet,
a national molecular typing scheme based on a standard method for PFGE;
a similar network is being set up for the major Salmonella reference laboratories
in Europe with research funding from the European Commission. Developing
compatible networks for the exchange of real-time molecular data for S.
enterica on an intercontinental scale would be of major benefit for
the global control of salmonellosis.
Ms. Lindsay is a research scientist completing a doctorate degree in
studies on antibiotic resistance in organisms from farm wastes. Her primary
research interests are in the molecular epidemiology of antibiotic resistance
in gram-negative bacteria.
References
- Outbreak of Salmonella typhimurium DT204b in
Britain and elsewhere in Europe. Commun Dis Rep 2000;10:349.
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phage type) infection: the Netherlands. Eurosurveillance Weekly 2000;4:000928.
Available from: URL: http://www.eurosurveillance.org
- Walker RA, Saunders N, Lawson AJ, Lindsay EA, Dassama M, Ward LR,
et al. Use
of a LightCycler gyrA mutation assay for rapid identification
of mutations conferring decreased susceptibility to ciprofloxacin in
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J Clin Microbiol 2001;39:1443–8.
- Walker RA, Lindsay E, Woodward MJ, Ward LR, Threlfall EJ.
Variation in clonality and antibiotic-resistance genes among multiresistant
Salmonella enterica serotype typhimurium phage-type U302 (MR
U302) from humans, animals and foods. Microb Drug Resist 2001;7:13–21.
- Powell NG, Threlfall EJ, Chart H, Rowe B. Subdivision
of Salmonella enteritidis PT4 by pulsed-field gel electrophoresis:
potential for epidemiological surveillance. FEMS Microbiol Lett
1994;119:193–8.
- Scott F, Threlfall EJ, Stanley J, Arnold C.
Fluorescent amplified fragment length polymorphism genotyping of Salmonella
Enteritidis: a method suitable for rapid outbreak recognition.
Clin Microbiol Infect 2001;7:479–85.
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