Quantitative Tracking of Salmonella Enteritidis Transmission Routes Using Barcode-Tagged Isogenic Strains in Chickens: Proof-of-Concept Study

Abstract

Salmonella is an important foodborne bacterial pathogen, however, a fundamental understanding on Salmonella transmission routes within a poultry flock remains unclear. In this study, a series of barcode-tagged strains were constructed by inserting six random nucleotides into a functionally neutral region on the chromosome of S. Enteritidis as a tool for quantitative tracking of Salmonella transmission in chickens. Six distinct barcode-tagged strains were used for infection or contamination at either low dose (10³ CFUs; three strains) or high dose (10⁵ CFUs; three strains) in three independent experiments (Experiment 1 oral gavage; Experiment 2 contaminated feed; Experiment 3 contaminated water). For all chick experiments, cecal and foot-wash samples were collected from a subset of the chickens at days 7 or/and 14, from which genomic DNA was extracted and used to amplify the barcode regions. After the resulting PCR amplicons were pooled and analyzed by MiSeq sequencing, a total of approximately 1.5 million reads containing the barcode sequences were analyzed to determine the relative frequency of every barcode-tagged strain in each sample. In Experiment 1, the high dose of oral infection was correlated with greater dominance of the strains in the ceca of the respective seeder chickens and also in the contact chickens yet at lesser degrees. When chicks were exposed to contaminated feed (Experiment 2) or water (Experiment 3), there were no clear patterns of the barcode-tagged strains in relation to the dosage, except that the strains introduced at low dose required a longer time to colonize the ceca with contaminated feed. Most foot-wash samples contained only one to three strains for the majority of the samples, suggesting potential existence of an unknown mechanism(s) for strain exclusion. These results demonstrated the proof of concept of using barcode tagged to investigate transmission dynamics of Salmonella in chickens in a quantitative manner.

Keywords: Salmonella Enteritidis; barcode-tagged isogenic strains; chickens; quantitative tracking; transmission.

Figure 1

Schematics of the construction of barcode-tagged Salmonella Enteritidis 13A strains.

Figure 2

Transmission of the Salmonella barcode-tagged strains in contact chickens in oral infection model. In Experiment 1, six seeder chickens were infected by different dose of SE barcode strains (BC1, BC2, and BC3 are used for infection of three chickens at 10³ CFUs; BC4, BC5, and BC6 are used for infection of other three chickens at 10⁵ CFUs). Other 10 chickens were roomed together with these six seeder chickens and named as contact chickens. Three contact chickens were euthanized on day 7, and four contact chickens were euthanized on day 14. The cecal tonsil and foot wash samples were collected from each chicken by aseptic technique. x-axis represents different contact chickens from Experiment 1, and y-axis represents different SE barcode strains. The number in bubble presents the relative abundance of each barcode strain in each chicken. Bigger size and red color means the higher relative abundance, and smaller size and blue color means lower relative abundance.

Figure 3

Transmission of the Salmonella barcode-tagged strains in feed contamination model. In Experiment 2, the feed was contaminated by six SE barcode strains at two doses (BC1, BC2, and BC3 were introduced into the feed at 10³ CFUs, and BC4, BC5, and BC6 were at 10⁵ CFUs on day 1). Two chickens were euthanized on day 7, and the other four chickens were euthanized on day 14. The cecal and foot wash samples were collected from each chicken by aseptic technique and used for isolation of genomic DNA. Following PCR and MiSeq analyses of barcode regions, the number of sequence reads corresponding to different barcodes were used to determine the relative abundance (%) of each SE barcode strain from each sample. x-axis represents different chickens from Experiment 2, and y-axis represents different SE barcode strains. Bigger size and red color means the higher relative abundance, and smaller size and blue color means lower relative abundance.

Figure 4

Transmission of the Salmonella barcode-tagged strains in water contamination model. In Experiment 3, the water was contaminated by 6 SE barcode strains at two doses (BC1, BC2, and BC3 were introduced into the water at 10³ CFUs, and BC4, BC5, and BC6 were at 10⁵ CFUs on day 1). Four chickens were euthanized on day 7, and the other four chickens were euthanized on day 14. The cecal and foot wash samples were collected from each chicken by aseptic technique and used for isolation of genomic DNA. Following PCR and MiSeq analyses of barcode regions, the number of sequence reads corresponding to different barcodes were used to determine the relative abundance (%) of each SE barcode strain from each sample. x-axis represents different chickens from Experiment 2, and y-axis represents different SE barcode strains. Bigger size and red color means the higher relative abundance, and smaller size and blue color means lower relative abundance.