Search

Genes prot6E and sefA are used as targets for detection of Salmonella enterica subsp. enterica serovar Enteritidis (Salmonella ser. Enteritidis). We investigated variations in these genes across 64 different Salmonella ser. Enteritidis strains isolated from egg and chicken samples, then used Whole Genome Sequence (WGS) data to model the structures of their protein products. Isolates were sequenced using Illumina technologies. Based on the resulting phylogenetic tree, our isolates clustered in 2 distinct clades. All isolates carried prot6E and sefA. Comparative genomic analyses indicated two non-synonymous mutations (Glycine → Serine and Valine → Isoleucine) of prot6E in 11 isolates (9 egg samples, 2 chicken samples). However, SWISS-MODEL was unable to clearly model the protein structure of these two mutations. We identified one non-synonymous mutation (Valine → Glutamic Acid) in the sefA gene in 4 isolates from egg samples. The model for the protein structure of this mutant gene was clearly different from that of the other isolates studied herein. Circular maps of plasmid genomes from two PacBio platform-sequenced Salmonella ser. Enteritidis isolates revealed prot6E gene was located on the tail of the plasmid. Based on the biosynthesis of amino acids - Reference pathway in the KEGG pathway Database, the transition of amino acid from sefA Var. was a transversion from essential amino acid to non-essential amino acid, while that of prot6E Var.1 happened between the conditionally non-essential amino acid, and prot6E Var. 2 occurred between essential amino acids. Properties of these mutated amino acids, such as side-chain polarity or charge, may contribute to the occurrence and rate of mutations in prot6E and sefA. These insights can be used to improve detection methods for Salmonella ser. Enteritidis.

With the increasing focus on the food safety, rapid methods for the detection of Salmonella are crucial for both food industry and regulatory agencies. Recently, many molecular methodologies with diverse technologies have been introduced. Roka Atlas® Salmonella Assay (SEN) is a molecular method that uses ribosomal RNA as target for detection, which is theoretically more sensitive than PCR or isothermal amplification methods that target the DNA sequences of single genes. In this study, SEN assay was compared with four PCR- and isothermal amplification-based assays and a culture method, such as the MicroSEQ® Salmonella spp. Detection kit (MicroSEQ), 3M™ Molecular Detection Assay (MDA) Salmonella, ANSRTM Salmonella Assay (ANSR), and Pro-AmpRTTM SALM spp. Kit (Pro-AmpRT). Food samples were prepared and analyzed according to the current U. S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Salmonella culture method. A total of 155 bacterial isolates (121 for Salmonella inclusivity and 34 for Salmeonlla exclusivity) and 200 egg product samples inoculated at a level of 1–5 CFU/25 g were analyzed. The study also estimated the limit of detection of these molecular methods, and illustrated their advantages and disadvantages. For exclusivity, all 34 non-Salmonella isolates were negative by all 5 molecular methods studied. For inclusivity, all 121 Salmonella isolates were positive by MDA, ANSR, and Pro-AmpRT methods. However, the SEN and MicroSEQ results were negative for 9 samples inoculated with Salmonella bongori. The detection limit of the 5 molecular methods ranged from 1.76 to 3.76 log CFU/mL pre-enrichment culture, with the SEN assay being the most sensitive (1.76 – 2.64 log CFU/mL). The results indicated that the SEN assay was as effective and sensitive in detecting Salmonella enterica in egg products as was the FDA BAM culture method and the 4 other isothermal amplification and PCR methods evaluated in the study.

Salmonella ser. Enteritidis is a major public health concern worldwide. Loop-mediated isothermal amplification (LAMP) is a novel simple, easy-to-operate detection technology that amplifies DNA with high speed, efficiency, and specificity under isothermal conditions. The objective of this study was to develop a new LAMP assay for the detection of Salmonella ser. Enteritidis. The Prot6E gene located on a virulence plasmid of Salmonella ser. Enteritidis, encoding fimbrial biosynthesis protein, was the target for detection. The primer set was designed by using Primer Explorer V4 software and evaluated for its effectiveness in detecting Salmonella ser. Enteritidis strains SE12 (PT14b), 18579 (PT4), and CDC_2010K_1441 (PT8) using isothermal master mix under an approximately 35 min reaction by Genie III instrument (OptiGene, UK). Ratio of outer and inner primers, amount of DNA template, reaction temperature and time were optimized. Inclusivity test using 114 Salmonella ser. Enteritidis strains showed 97.4% positive for prot6E gene. For exclusivity testing, 34 non-Salmonella ser. Enteritidis Salmonella strains (27 serotypes) and 35 non-Salmonella strains (14 species) were tested and they were 100% negative. Results from the LAMP assay on 200 samples from egg products inoculated with 1–5 CFU/25 g completely matched with that from culture method and real-time PCR, and less than 1.2–12 CFU per reaction Salmonella ser. Enteritidis could be detected. This LAMP method can be of high value to the food industry due to its various advantages such as speed, specificity, sensitivity, cost- and labor-efficiency.

Listeriosis is an invasive illness typically caused by the ingestion of foods contaminated with Listeria monocytogenes. In 2015, an outbreak of listeriosis was linked to ice cream products produced on a specific production line at Facility X. The United States Food and Drug Administration (FDA) obtained samples representing several lots of three products manufactured on that line from May 2014 through January 2015. Two of these products, A and B, while not linked to any reported illnesses, were analyzed to determine the frequency of contamination and the contamination level for risk assessment and dose-response analyses. These enumerations were performed utilizing a Most Probable Number (MPN) method, with a lower detection limit of 0.03 MPN/g, on 344 samples of Product A and 95 samples of Product B. Ten lots of Product A were analyzed and 77% of the samples tested were found to be positive for L. monocytogenes. Five lots of Product B were analyzed and 46% of the tested samples were found to be positive. Additionally, the level of contamination of positive Product B samples was always less than 1 MPN/g. The contamination levels of both products, overall, were low with median values of 0.1 MPN/g and 0.02 MPN/g for Products A and B, respectively. A majority of Product A samples (52%) were contaminated at levels of less than 1 MPN/g and only one sample was above 100 MPN/g. Whole genome sequencing (WGS) of L. monocytogenes isolated from ice cream samples produced in the line suggested minor strain differences related to product type, possibly due to differences in the food matrices and/or differences in the manufacturing equipment. Overall, the data showed a consistent low level of contamination in products produced from a single production line over a nine month period.