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During the months of June and July 1996, a sudden increase in the number of cases involving Salmonella group D was observed in a suburb west of Boston, Mass. Investigation by state public health epidemiologists linked these cases of gastroenteritis to a single restaurant. Stool samples and/or subcultures from ill patrons and food handlers, as well as a portion of a chicken sandwich that was partially consumed by one of the ill patrons, were submitted to the Massachusetts State Laboratory Institute for culture, identification or confirmation, serotyping, and strain typing. Salmonella javiana was isolated from 66 of the 95 clinical specimens submitted, representing 55 patrons and 11 restaurant employees. In addition, S. javiana was isolated from the leftover chicken sandwich.

In the Commonwealth of Massachusetts, S. javiana is a rarely encountered organism. The average reported incidence of S. javiana for the 5 years preceding this outbreak was 15.2/year. In the 3-week period during the outbreak, the total number of S. javiana isolates received increased dramatically to 75.

Laboratory and epidemiological methods used previously to establish chains of transmission in food-borne infections have been limited to serotyping (subtyping) and classical epidemiological case studies, all of which are not sufficiently sensitive to distinguishing individual strains. The application of pulsed-field gel electrophoresis (PFGE) provides precise information that can be used to accept or reject epidemiologic associations to a high degree of confidence (4-6). PFGE is believed to possess a discriminating capacity greater than those of ribotyping and other probe-based restriction fragment length polymorphism methods (7). We report here the first use of PFGE to confirm the chain of transmission of S. javiana from restaurant food handlers or food to patrons.

Bacterial strains. Sixty-seven isolates of S. javiana were isolated from specimens from 55 ill restaurant patrons, 11 food handlers, and one chicken sandwich that had been partially consumed by one of the ill patrons. The chicken sandwich isolate was used as a control strain that was included on each gel. Included were 13 non-outbreak-related S. javiana strains which were temporally and/or geographically different from that involved in the outbreak. A slight modification of standard serotyping methods was used to confirm the positive isolates as S. javiana (2). All isolates were stored on nutrient agar slants at room temperature.

DNA preparation. Intact genomic DNA was prepared by a modification of the method described by Barrett et al. (1). Briefly, single-colony isolates were selected from purity blood agar plates and grown at 37°C in Luria-Bertani nutrient broth to mid-log phase. One milliliter of suspension was harvested, centrifuged, washed with 1 ml of SE wash buffer, and then resuspended in 0.5 ml of SE wash buffer. An equal volume of 1.2% plug agarose (1.2% Bio-Rad chromosomal grade agarose, 10 mM Tris-HCl [pH 7.4], 0.1 mM EDTA [pH 8.0]) was added to the suspension, and the mixture was injected into molds to form plugs. The agarose plugs were incubated in lysis solution containing 1.0 mg of proteinase K per ml in a 50°C water bath overnight. The proteinase K was then removed from the plugs by four washes in 5 ml of Tris-EDTA buffer. DNA plugs can then be stored in Tris-EDTA buffer at 4°C.

Restriction endonuclease digestion and PFGE. A portion (1 by 3.5 by 5 mm) of each plug was cut and washed in restriction buffer. The slices were digested overnight with selected restriction enzymes. The enzyme concentrations, buffers, and incubation periods used were those recommended by the manufacturer. Selection of restriction enzymes was based on the recognition sites of the enzymes XbaI (5'-TCTAGA-3') and SpeI (5'-ACTAGT-3') (New England Biolabs, Inc., Beverly, Mass.) and the 50 to 54% G+C content of previously reported Salmonella species (3). Restriction fragments of DNA were separated by PFGE with the CHEF-Mapper apparatus (Bio-Rad) through a 1.0% gel (1.0% Bio-Rad pulsed-field-certified agarose, 0.5× Tris-borate-EDTA buffer). Electrophoresis was performed over a period of 22 h (initial switch time, 2.16 s; final switch time, 54.17 s) at 6 V/cm and 14°C in 0.5× Tris-borate-EDTA buffer. A lambda ladder standard (Bio-Rad) was used as a molecular size marker. The gels were stained with ethidium bromide and then illuminated on a UV transilluminator (302 nm; Bio-Rad). Results of the XbaI restriction were confirmed by using the second enzyme SpeI. Isolates were considered to be genetically indistinguishable only if their restriction pattern profiles had the same numbers of bands and the corresponding bands were of the same apparent sizes when both restriction enzymes were used.

DNA analysis. DNA fragment patterns were electronically scanned directly into a computer via a charge-coupled device video camera directly connected to a personal computer. Once the images had been acquired, they were compared both visually and with the assistance of analysis software (Molecular Analyst Fingerprinting Plus by Bio-Rad) to determine relatedness.

View larger version (122K): [in this window] [in a new window]   FIG. 1.   PFGE fingerprints of S. javiana isolates from a suspected outbreak after digestion with XbaI. Lanes: 1, 8, and 15, lambda molecular size marker ladder (Bio-Rad); 4 and 5, unrelated S. javiana isolates; 2, isolate from the leftover chicken sandwich (control strain); 3 and 11 to 14, representative sampling of results for all patrons; 6, 7, 9 and 10, isolates from food handlers.

View larger version (120K): [in this window] [in a new window]   FIG. 2.   PFGE fingerprints of S. javiana isolates from a suspected outbreak after digestion with SpeI. Lanes: 1, 8, and 15, lambda molecular size marker ladder (Bio-Rad); 4 and 5, unrelated S. javiana isolates; 2, isolate from the leftover chicken sandwich (control strain); 3 and 11 to 14, representative sampling of results for all patrons; 6, 7, 9, and 10, isolates from food handlers.

Discussion. This report describes an outbreak investigation whereby identification of the implicated organism by PFGE analysis of DNA content allowed us to trace a genetically indistinguishable strain of S. javiana from food handlers to leftover food and finally to patrons who were epidemiologically linked to the outbreak.