Overview of S. Dublin genomes in study

Geographical distribution and clonal relationship of 1,303 S. Dublin genomes in the study. A) Number of S. Dublin genomes according to geographical region and time interval of collection. B) Distribution of human, animal and food S. Dublin genomes collected over time. C) Distribution of S. Dublin sequence types (ST) from each region. Each coloured circle correlates to an ST. Circles in green indicate no ST was identified. D) Unrooted phylogenetic tree of 1,303 S. Dublin genomes. Two main lineages of ST74 (n=8) and ST10 (n=1,295) shown in blue and pink respectively. E) Relatedness of known sequence types (STs) to ST10. The nodes are labelled with the ST and the branch lengths are indicative of the number of dissimilar locus variants to ST10.

Estimation of divergence time

Maximum clade credibility (MCC) tree of the 667 S. Dublin ST10 genomes. The time in years is shown on the x-axis. From left to right, the coloured heatmaps refer to the region of origin, the susceptibility profile, the BAPs clade, and the presence of IncC and IncN plasmid types.

Resistance profiles of S. Dublin genomes

Observed resistance profiles of S. Dublin genomes spanning three-time intervals for each region. A) Frequency of susceptible and resistant S. Dublin genomes from each time period B) Antimicrobial resistance profiles observed spanning three time periods. The colour of the circle corresponds to an antimicrobial class, while the height is relative to the number of genomes. B) Heavy metal and biocide resistance profiles observed. The colour of the circle corresponds to its respective heavy metal/biocide class, while the height is relative to the number of genomes.

Invasive factors associated with S. Dublin genomes

Correlation of Salmonella pathogenicity islands (SPIs) to S. Dublin’s invasiveness. A) Validation of the likelihood of invasiveness prediction tool using a variety of publicly available genomes consisting of non-invasive and invasive Salmonella serovars (Supplementary Table 8). B) The correlation of index scores to the source and its sequence type based on the region. * indicates a statistically significant p-value between the index scores of two sources. C) Chromosomal assemblies of ST10 (blue) and ST74 (pink) genomes aligned to S. Dublin chromosomal reference CT_02021853 (CP001144.1) with a lower limit of 80% identity D) Aligned sequence of SPI-6 region of an ST10, AUSMDU00056868, and an ST74, AUSMDU00016676, in comparison to SPI-6 region of reference CT_02021853. E) Aligned sequence of SPI-19 region of an ST10, AUSMDU00056868, and an ST74, AUSMDU00016676 in comparison to SPI-19 region of reference CT_02021853. SPI, Salmonella pathogenicity island.

S. Dublin ST74 isolates demonstrate increased intracellular replication in human macrophages over ST10 isolates

Differentiated THP-1 (human monocyte) cells were infected at an MOI:10 with selected ST10 and ST74 S. Dublin isolates. A, B) THP-1 cells were lysed, and intracellular bacteria enumerated as fold change in replication and C-F) CFU/well at time 0, 3, 9 and 24 hpi. For A), each measurement at times 3, 9 and 24 hpi represents the fold change in CFU/well compared to 0 hpi, the start of infection, recorded as a biological replicate (performed in technical triplicate), with error bars indicating ± 1 standard deviation of n = 3 biological replicates. For C-D), each dot represents CFU/well of a biological replicate (performed in technical triplicate), with error bars indicating ± 1 standard deviation of n = 3 biological replicates. Statistical significance was determined by nested student’s t-test. MOI, multiplicity of infection, hpi, hours post-infection, CFU, colony-forming units, FC, fold-change.

S. Dublin ST74 isolates induce comparable cytotoxicity to ST10 isolates in human macrophages, despite increased intracellular growth

A-C) Cell supernatants were assayed for LDH as a measure of cytopathic effects of infection, and % cytotoxicity was calculated by comparison with 100% lysed uninfected control cells. Samples were collected from THP-1 cells at 3, 9 and 24 hpi. Each point represents the % cytotoxicity of a biological replicate (performed in technical triplicate), with error bars indicating ± 1 standard deviation of n = 3 biological replicates. Statistical significance was determined by nested student’s t-test. D) IL-1ß secretion from THP-1 cells infected for 9 hours with S. Dublin isolates. Error bars indicate ± 1 standard deviation of n = 3 biological replicates and statistical significance determined by student’s t-test E) Immunoblot of cleaved IL-1ß (Asp166) in THP-1 cell lysates infected with S. Typhimurium reference strain SL1344, S. Dublin ST10 (AUSMDU00016673), or S. Dublin ST74 (AUSMDU00016682) over 0, 3, 8 and 24 hours. F) PCA plot comparing replication rate, % cytotoxicity and IL-1β secretion from THP-1 cells infected with S.Dublin isolates at 9 hpi. Each dot represents each biological replicate of three (averaged from three technical replicates). .MOI, multiplicity of infection, hpi, hours post-infection, CFU, colony-forming units, LDH, lactate dehydrogenase.