Ecological prevalence, genetic diversity, and epidemiological aspects of Salmonella isolated from tomato agricultural regions of the Virginia Eastern Shore

Abstract

Virginia is the third largest producer of fresh-market tomatoes in the United States. Tomatoes grown along the eastern shore of Virginia are implicated almost yearly in Salmonella illnesses. Traceback implicates contamination occurring in the pre-harvest environment. To get a better understanding of the ecological niches of Salmonella in the tomato agricultural environment, a 2-year study was undertaken at a regional agricultural research farm in Virginia. Environmental samples, including tomato (fruit, blossoms, and leaves), irrigation water, surface water and sediment, were collected over the growing season. These samples were analyzed for the presence of Salmonella using modified FDA-BAM methods. Molecular assays were used to screen the samples. Over 1500 samples were tested. Seventy-five samples tested positive for Salmonella yielding over 230 isolates. The most commonly isolated serovars were S. Newport and S. Javiana with pulsed-field gel electrophoresis yielding 39 different patterns. Genetic diversity was further underscored among many other serotypes, which showed multiple PFGE subtypes. Whole genome sequencing (WGS) of several S. Newport isolates collected in 2010 compared to clinical isolates associated with tomato consumption showed very few single nucleotide differences between environmental isolates and clinical isolates suggesting a source link to Salmonella contaminated tomatoes. Nearly all isolates collected during two growing seasons of surveillance were obtained from surface water and sediment sources pointing to these sites as long-term reservoirs for persistent and endemic contamination of this environment.

Keywords: Salmonella Newport; environmental reservoirs; epidemiological impact; prevalence and diversity; tomatoes.

Figure 1

VES sampling sites. Map of Virginia Eastern Shore with locations of: various VES waterways (A–F) and AREC (G).

Figure 2

Prevalence of Salmonella in VES. Bar and line graph showing prevlance and number of positive samples (respectively) for each month tested. A total of 1570 samples were screened for the presence of Salmonella. Overall, 8.4% of the 892 cultured samples were positive for Salmonella.

Figure 3

Serotype distribution of the 234 isolates collected and analyzed, to date, from VES during the summers 2009–2011. Serotypes were determined by molecular serotyping, inferred from PFGE analysis, or from traditional serotyping. Twenty-one different serotypes were found. S. Newport was the most prominent serotype, followed closely by S. Javiana.

Figure 4

PFGE analysis. PFGE was performed on 234 isolates from VES collected during the summers of 2009–2011. PFGE was executed as per standard CDC-PulseNet protocols using XbaI. Shown is one representative pattern from isolates with the same pattern along with internal pattern number. Each representative pattern was submitted to PulseNet to obtain an official pattern name, if available. The distribution of each pattern is shown. Many serotypes show more than one PFGE pattern subtype, for example S. Javiana has seven different PFGE pattern subtypes.

Figure 5

Whole genome sequence analysis of 19 S. Newport strains. Strains included four isolated from the summer 2010, nine historical tomato farm isolates from the VES that are part of CFSAN's culture collection, and six clinical isolates (kindly provided by the Departments of Public Health in Maryland, Virginia and Washington D.C.) from a cluster of illnesses caused by S. Newport in the summer of 2010. Tree was built using GARLI with 1000 bootstrap replicates from a SNP matrix (see Materials and Methods). Two well-supported clades are seen (1 and 2). Clade 1 consists of historical tomato farm isolates and two clinical isolates. Clade 2 consists of historical tomato farm isolates, four VES AREC isolates from 2010 and four clinical isolates. Two AREC isolates, one from goose feces (CFSAN000929) and one from creek sediment (CFSAN000927) (in Clade 2a) have the fewest number of SNP differences from the clinical isolates (CFSAN000859, 000860, 000864, 000861).

Figure 6

SNP profile of 19 S. Newport strains. Graphic of SNP position, across the chromosome of S. Newport strain SL254, from matrix used to construct the WGS tree (Figure 4). The number of isolates containing the SNP at each position presented on the y-axis. All 19 strains shared 30903 out of 33301 SNPs. All strains in Clade 1 share 671 out of 680 SNPs. All strains in Clade 2 share 288 out of 299 SNPs.