Genomic Evidence of Environmental and Resident Salmonella Senftenberg and Montevideo Contamination in the Pistachio Supply-chain

Pistachios have been implicated in two salmonellosis outbreaks and multiple recalls in the U.S. This study performed a retrospective data analysis of Salmonella associated with pistachios and a storage study to evaluate the survivability of Salmonella on inoculated inshell pistachios to further understand the genetics and microbiological dynamics of this commodity-pathogen pair. The retrospective data analysis on isolates associated with pistachios was performed from both short-read and long-read sequencing technologies. The sequence data were analyzed using the FDAs Center for Food Safety and Applied Nutrition Single Nucleotide Polymorphism (SNP) analysis and Whole Genome Multi-locus Sequence Typing (wgMLST) pipeline. The storage study evaluated the survival of five strains of Salmonella on pistachios, both in a cocktail as well as individually. Our results demonstrate: i) evidence of persistent Salmonella Senftenberg and Salmonella Montevideo strains in pistachio environments, some of which may be due to clonal resident strains and some of which may be due to preharvest contamination; ii) presence of the Copper Homeostasis and Silver Resistance Island (CHASRI) in Salmonella Senftenberg and Montevideo strains in the pistachio supply chain; and iii) different serovars of Salmonella enterica, including Salmonella Senftenberg and Salmonella Montevideo, are able to survive in pistachios over an extended period of time. ImportancePistachios have been linked to multistate outbreaks caused by Salmonella serovar Senftenberg (2013, 2016) and serovar Montevideo (2016). This comprehensive study of whole-genome-sequence (WGS) data from Senftenberg and Montevideo isolates associated with pistachio outbreaks, recalls, and investigations over a nine-year period (2009-2018) was conducted to provide insights into evolutionary relationships and persistence. Multiple analytical approaches were applied including MLST, wgMLST and phylogenetic SNP analysis. The CHASRI was identified in all isolates associated with the sequence type 14 (ST14) and ST316, suggesting an adaptation in response to extrinsic pressures, such as in the farm environment. There is evidence of persistent Salmonella Senftenberg and Montevideo strains in pistachio pre-harvest and post-harvest environments. Utilizing metagenomic sequencing during an inoculated storage study of pistachios allowed for determination of surviving serovars over an extended period of time. Defining the mechanisms of persistence of these strains is of high importance to public health.