Genomic evidence reveals numerous Salmonella enterica serovar Newport reintroduction events in Suwannee watershed irrigation ponds

Abstract

Our previous work indicated a predominance (56.8%) of Salmonella enterica serovar Newport among isolates recovered from irrigation ponds used in produce farms over a 2-year period (B. Li et al., Appl Environ Microbiol 80:6355-6365, http://dx.doi.org/10.1128/AEM.02063-14). This observation provided a valuable set of metrics to explore an underaddressed issue of environmental survival of Salmonella by DNA microarray. Microarray analysis correctly identified all the isolates (n = 53) and differentiated the S. Newport isolates into two phylogenetic lineages (S. Newport II and S. Newport III). Serovar distribution analysis showed no instances where the same serovar was recovered from a pond for more than a month. Furthermore, during the study, numerous isolates with an indistinguishable genotype were recovered from different ponds as far as 180 km apart for time intervals as long as 2 years. Although isolates within either lineage were phylogenetically related as determined by microarray analysis, subtle genotypic differences were detected within the lineages, suggesting that isolates in either lineage could have come from several unique hosts. For example, strains in four different subgroups (A, B, C, and D) possessed an indistinguishable genotype within their subgroups as measured by gene differences, suggesting that strains in each subgroup shared a common host. Based on this comparative genomic evidence and the spatial and temporal factors, we speculated that the presence of Salmonella in the ponds was likely due to numerous punctuated reintroduction events associated with several different but common hosts in the environment. These findings may have implications for the development of strategies for efficient and safe irrigation to minimize the risk of Salmonella outbreaks associated with fresh produce.

FIG 1

Probe set design used in the microarray.

FIG 2

Microarray scatter plots of pairwise comparisons demonstrating gene-level differences between strains analyzed in this study. (A to C) Three pairs of isolates C110 (S. Newport) (A), C151 (S. Newport) (B), and C180 (S. Enteritidis) (C), each of which was recovered from the same irrigation pond, were compared. (D to F) S. Newport isolate C75 was compared with S. Newport isolate C177 (recovered from a different pond) (D), S. Newport isolate SL1511 (recovered from recent outbreaks) (E), and strain SARB36 (F).

FIG 3

Hierarchical clustering of RMA-summarized microarray data employing a database of over 760 Salmonella reference strains and outbreak strains from DMB, FDA, collections integrated with 56 isolates analyzed in this study. The resulting dendrogram represents a large comprehensive phylogenic tree which can be viewed in Fig. S2 in the supplemental material. Using the same strategy, a streamlined cluster dendrogram was generated among the 56 isolates analyzed by microarray analysis.

FIG 4

Microarray scatter plots of pairwise comparisons demonstrating gene-level differences between strains analyzed in this study. Four S. Newport II isolates C84, C124, C126, and C234, were recovered from different ponds at different sample dates. (A to C) The genotype of C84 was compared with those of C124 (A), C126 (B), and C234 (C). (D to F) The genotype of C84 was also compared with those of two S. Newport II isolates, C110 (D) and C83 (E), and S. Newport II strain C75 (F).

FIG 5

Conceptual model for Salmonella transmission through surface water, showing microbial interactions and intersections between the environment, animals, and humans that influence the dynamic nature of the Salmonella population within an irrigation pond ecology through which the consumption of produce contaminated with irrigation water leads to human illness.