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Our previous study demonstrated that whole genome sequencing (WGS) data generated by Oxford Nanopore Technologies (ONT) can be used for rapid and accurate prediction of selected Salmonella serotypes. However, one limitation is that established methods for WGS-based serotype prediction, utilizing data from either ONT or Illumina, cannot differentiate certain serotypes and serotype variants with the same or closely related antigenic formulae. This study aimed to evaluate nanopore sequencing and additional data analysis for identification of Salmonella enterica Choleraesuis var. Kunzendorf and S. enterica Orion var. 15

Our previous study demonstrated that whole genome sequencing (WGS) data generated by Oxford Nanopore Technologies (ONT) can be used for rapid and accurate prediction of selected Salmonella serotypes. However, one limitation is that established methods for WGS-based serotype prediction, utilizing data from either ONT or Illumina, cannot differentiate certain serotypes and serotype variants with the same or closely related antigenic formulae. This study aimed to evaluate nanopore sequencing and additional data analysis for identification of Salmonella enterica Choleraesuis var. Kunzendorf and S. enterica Orion var. 15+, 34+, thus overcoming this limitation. Five workflows that combined different flow cells, library construction methods and basecaller models were evaluated and compared. The workflow that consisted of the R9 flow cell, rapid sequencing library construction kit and guppy basecaller with base modified model performed best for Single Nucleotide Polymorphism (SNP) analysis. With this workflow, 99.98% of matching identity between assembled genomes from ONT and that from Illumina was achieved. Less than five high-quality SNPs differed when comparing sequencing data between ONT and Illumina. SNP typing successfully identified Choleraesuis var. Kunzendorf. While prophage prediction further differentiated Orion var. 15+, 34+ from the other two Orion variants. Our study improves the readiness of ONT as a Salmonella subtyping and source tracking tool for food industry applications.

Internalization of human pathogens, common in many types of fresh produce, is a threat to human health since the internalized pathogens cannot be fully inactivated/removed by washing with water or sanitizers. Given that pathogen internalization can be affected by many environmental factors, this study was conducted to investigate the influence of two types of plant stress on the internalization of Salmonella Typhimurium in iceberg lettuce during pre-harvest. The stresses were: abiotic (water stress induced by extreme weather events) and biotic (phytopathogen infection by lettuce mosaic virus [LMV]). Lettuce with and without LMV infection were purposefully contaminated with green fluorescence protein-labeled S. Typhimurium on the leaf surfaces. Lettuce was also subjected to water stress conditions (drought and storm) which were simulated by irrigating with different amounts of water. The internalized S. Typhimurium in the different parts of the lettuce were quantified by plate count and real-time quantitative PCR and confirmed with a laser scanning confocal microscope. Salmonella internalization occurred under the conditions outlined above; however internalization levels were not significantly affected by water stress alone. In contrast, the extent of culturable S. Typhimurium internalized in the leafy part of the lettuce decreased when infected with LMV under water stress conditions and contaminated with high levels of S. Typhimurium. On the other hand, LMV-infected lettuce showed a significant increase in the levels of culturable bacteria in the roots. In conclusion, internalization was observed under all experimental conditions when the lettuce surface was contaminated with S. Typhimurium. However, the extent of internalization was only affected by water stress when lettuce was infected with LMV.