Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
The manuscript consists of two separate but interlinked investigations: genomic epidemiology and virulence assessment of Salmonella Dublin. ST10 dominates the epidemiological landscape of S. Dublin, while ST74 was uncommonly isolated. Detailed genomic epidemiology of ST10 unfolded the evolutionary history of this common genotype, highlighting clonal expansions linked to each distinct geography. Notably, North American ST10 was associated with more antimicrobial resistance compared to others. The authors also performed long-read sequencing on a subset of isolates (ST10 and ST74) and uncovered a novel recombinant virulence plasmid in ST10 (IncX1/IncFII/IncN). Separately, the authors performed cell invasion and cytotoxicity assays on the two S. Dublin genotypes, showing differential responses between the two STs. ST74 replicates better intracellularly in macrophages compared to ST10, but both STs induced comparable cytotoxicity levels.
Comparative genomic analyses between the two genotypes showed certain genetic content unique to each genotype, but no further analyses were conducted to investigate which genetic factors were likely associated with the observed differences. The study provides a comprehensive and novel understanding of the evolution and adaptation of two S. Dublin genotypes, which can inform public health measures.
The methodology included in both approaches was sound and written in sufficient detail, and data analysis was performed with rigour. Source data were fully presented and accessible to readers. Certain aspects of the manuscript could be clarified and extended to improve the manuscript.
(1) For epidemiology purposes, it is not clear which human diseases were associated with the genomes included in this manuscript. This is important since S. Dublin can cause invasive bloodstream infections in humans. While such information may be unavailable for public sequences, this should be detailed for the 53 isolates sequenced for this study, especially for isolates selected to perform experiments in vitro.
Thank you for the suggestion. We have added the sample type for the 53 isolates sequenced for this study. These additional details have been added to Supplementary Tables 1, 4, 9 and 10.
(2) The major AMR plasmid in described S. Dublin was the IncC associated with clonal expansion in North America. While this plasmid is not found in the Australian isolates sequenced in this study, the reviewer finds that it is still important to include its characterization, since it carries blaCMY-2 and was sustainedly inherited in ST10 clade 5. If the plasmid structure is already published, the authors should include the accession number in the Main Results.
We have provided accessions and context for two of the IncC hybrid plasmids that have been previously reported in the literature in the Introduction. The text now reads:
“These MDR S. Dublin isolates all type as sequence type 10 (ST10), and the AMR determinants have been demonstrated to be carried on an IncC plasmid that has recombined with a virulence plasmid encoding the spvRABCD operon (12,16,18,19). This has resulted in hybrid virulence and AMR plasmids circulating in North America including a 329kb megaplasmid with IncX1, IncFIA, IncFIB, and IncFII replicons (isolate CVM22429, NCBI accession CP032397.1) (12,16) and a smaller hybrid plasmid 172,265 bases in size with an IncX1 replicon (isolate N13-01125, NCBI accession KX815983.1) (19).”
Further characterisation of the IncA/C plasmid circulating in North America was beyond the scope of this study.
(a) The reviewer is concerned that the multiple annotations missing in plasmid structures in Supplementary Figures 5 & 6, and genetic content unique to ST10 and ST74 was due to insufficient annotation by Prokka. I would recommend the authors use another annotation tool, such as Bakta (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8743544/) for plasmid annotation, and reconstruction of the pangenome described in Supplementary Figure 10. Since the recombinant virulence plasmid in ST10 is a novel one, I would recommend putting Supplementary Figure 5 as a main figure, with better annotations to show the virulence region, plasmid maintenance/replication, and possible conjugation cluster.
In the supplementary figures of the plasmids, we sought to highlight key traits on interest on the plasmids, namely plasmid replicons, antimicrobial resistance and heavy metal resistance (Supplementary Figure 5) and virulence genes (Supplementary Figure 6). The inclusion of the accessions of publicly available isolates provide for characterised plasmids such as the S. Dublin virulence plasmid (NCBI accession: CP001143).
For the potentially hybrid plasmid with IncN/IncX1/IncFII reported in Supplementary Figure 6, we have undertaken additional analyses of the two Australian isolates to reannotate these isolates with Bakta which provides for more detailed annotations.
We have added new text to the methods which reads as:
“The final genome assemblies were confirmed as S. Dublin using SISTR and annotated using both Prokka v1.14.6 (69) for consistency with the draft genome assemblies and Bakta v1.10.1 (93) which provides for more detailed annotations (Supplementary Table 13). Both Prokka and Bakta annotations were in agreement for AMR, HMR and virulence genes, with Bakta annotating between 3-7 additional CDS which were largely ‘hypothetical protein’.”
For the pangenome analysis of the seven ST74 and ten ST10 isolates, we have continued to use the Prokka annotated draft genome assemblies for input to Panaroo.
(4) The authors are lauded for the use of multiple strains of ST10 and ST74 in the in vitro experiment. While results for ST74 were more consistent, readouts from ST10 were more heterogenous (Figure 5, 6). This is interesting as the tested ST10 were mostly clade 1, so ST10 was, as expected, of lower genetic diversity compared to tested ST74 (partly shown in Figure 1D. Could the authors confirm this by constructing an SNP table separately for tested ST10 and ST74? Additionally, the tested ST10 did not represent the phylogenetic diversity of the global epidemiology, and this limitation should be reflected in the Discussion.
In response to the reviewer’s comments, we have provided a detailed SNP table (Supplementary Table 12) to further clarify the genetic diversity within the tested ST10 and ST74 strains.
Additionally, we have expanded on the limitation regarding the phylogenetic diversity of the ST10 isolates in the Discussion, highlighting how the strains used in the in vitro experiments may not fully represent the global epidemiological diversity of S. Dublin ST10. The new text now reads:
“This study has limitations, including a focus on ST10 isolates from clade 1, which do not represent global phylogenetic diversity. Nonetheless, our pangenome analysis identified >900 uncharacterised genes unique to ST74, offering potential targets for future research. Another limitation is the geographic bias in available genomes, with underrepresentation from Asia and South America. This reflects broader disparities in genomic research resources but may improve as public health genomics capacity expands globally.”
(5) The comparative genomics between ST10 and ST74 can be further improved to allow more interpretation of the experiments. Why were only SPI-1, 2, 6, and 19 included in the search for virulome, how about other SPIs? ST74 lacks SPI-19 and has truncated SPI-6, so what would explain the larger genome size of ST74? Have the authors screened for other SPIs using more well-annotated databases or references (S. Typhi CT18 or S. Typhimurium ST313)? The mismatching between in silico prediction of invasiveness and phenotypes also warrants a brief discussion, perhaps linked to bigger ST74 genome size (as intracellular lifestyle is usually linked with genome degradation).
Systematic screening for SPIs with detailed reporting on individual genes and known effectors is still an area of development in Salmonella comparative genomics. In our characterisation of the virulome in this S. Dublin dataset we decided to focus on SPI1, SPI-2, SPI-6 and SPI-19 as these had been identified in previous studies and were considered to be most likely linked to the invasive phenotype of S. Dublin. We thought the truncation of SPI-6 and lack of SPI-19 in ST74 compared to the ST10 isolates would provide a basis to explore genomic differences in the two genotypes, with the screening for individual genes on each SPIs reported in Supplementary Figure 7 and Supplementary Table 9.
We have expanded upon the mismatching of the in silico prediction of invasiveness and phenotypes in the Discussion. We now explore the increased genome size and intracellular replication of the ST74 population. We hypothesise that invasiveness has not been studied as thoroughly in zoonotic iNTS as much as human adapted iNTS and S. Typhi, and the increased genome content may be required for survival in different host species. The new text now reads:
“Our phenotypic data demonstrated a striking difference in replication dynamics between ST10 and ST74 populations in human macrophages. ST74 isolates replicated significantly over 24 hours, whereas ST10 isolates were rapidly cleared after 9 hours of infection. ST74 induced significantly less host cell death during the early-mid stage of macrophage infection, supported by limited processing and release of IL-1ß at 9 hpi. While NTS are generally potent inflammasome activators (60), most supporting data come from laboratory-adapted S. Typhimurium strains. Our findings suggest that ST74 isolates may employ immune evasion mechanisms to avoid host recognition and activation of cell death signaling in early infection stages. Similar trends have been observed with S. Typhimurium ST313, which induces less inflammasome activation than ST19 during murine macrophage infection (61). This could facilitate increased replication and dissemination at later stages of infection. Consistent with this, we observed comparable cytotoxicity between ST10 and ST74 isolates at 24 hpi, suggesting ST74 induces cell death via alternative mechanisms once intracellular bacterial numbers are unsustainable. Further research is needed to identify genomic factors underpinning these observations.”
(6) On the epidemiology scale, ST10 is more successful, perhaps due to its ongoing adaptation to replication inside GI epithelial cells, favouring shedding. ST74 may tend to cause more invasive disease and less transmission via fecal shedding. The presence of T6SS in ST10 also can benefit its competition with other gut commensals, overcoming gut colonization resistance. The reviewer thinks that these details should be more clearly rephrased in the Discussion, as the results highly suggested different adaptations of two genotypes of the same serovar, leading to different epidemiological success.
We thank the reviewer for highlighting that we could rephrase this important point. We have added additional text in the Discussion to better interpret the differences in the two genotypes of S. Dublin and how this relates to difference epidemiological success. The new text now reads:
“While machine learning predicted lower invasiveness for ST74 compared to ST10, the increased genomic content of ST74 may support higher replication in macrophages. We speculate that increased intracellular replication could enhance systemic dissemination, though this requires in vivo validation. Invasiveness of S. enterica is often linked to genome degradation (4,62–64). However, this is mostly based on studies of human-adapted iNTS (ST313) and S. Typhi, leaving open the possibility that the additional genomic content of ST74 supports survival in diverse host species. An uncharacterised virulence factor may underlie this replication advantage. Collectively, these findings highlight phenotypic differences between S. Dublin populations ST10 and ST74. Enhanced intra-macrophage survival of ST74 could promote invasive disease, whereas the prevalence of ST10 may relate to better intestinal adaptation and enhanced faecal shedding. In vivo models are needed to test this hypothesis. Interestingly, the absence of SPI-19 in ST74, which encodes a T6SS, may reflect adaptation to enhanced replication in macrophages. SPI-19 has been linked to intestinal colonisation in poultry (23,56) and mucosal virulence in mice (56). It’s possible that the efficient replication of ST74 in macrophages might compensate for the absence of SPI-19, relying instead on phagocyte uptake via M cells or dendritic cells. The larger pangenome of ST74 compared to ST10 could further enhance survival within hosts. These findings highlight important knowledge gaps in zoonotic NTS host-pathogen interactions and drivers of emerging invasive NTS lineages with broad host ranges.”
Reviewer #2 (Public review):
This is a comprehensive analysis of Salmonella Dublin genomes that offers insights into the global spread of this pathogen and region-specific traits that are important to understanding its evolution. The phenotyping of isolates of ST10 and ST74 also offers insights into the variability that can be seen in S. Dublin, which is also seen in other Salmonella serovars, and reminds the field that it is important to look beyond lab-adapted strains to truly understand these pathogens. This is a valuable contribution to the field. The only limitation, which the authors also acknowledge, is the bias towards S. Dublin genomes from high-income settings. However, there is no selection bias; this is simply a consequence of publically available sequences.
Reviewer #1 (Recommendations for the authors):
(1) The Abstract did not summarize the main findings of the study. The authors should rewrite to highlight the key findings in genomic epidemiology (low AMR generally, novel plasmid of which Inc type, etc.) and the in vitro experiments. The findings clearly illustrate the differing adaptations of the two genotypes. Suggest to omit 'economic burden' and 'livestock' as this study did not specifically address them.
We agree with the Reviewer and have re-written the abstract to directly reflect the major outcomes of the research. We have also deleted wording such as ‘livestock’, ‘economic burden’ and ‘One Health’ as we did not specifically address these issues as highlighted by the Reviewer.
(2) Figure 2: The MCC tree should include posterior support in major internal nodes. The current colour scheme is also confusing to readers (columns 1, 2). Suggest to revise and include additional key information as columns: major AMR genes (blaCMY-2, strAB, floR) and mer locus, so this info can be visualized in the main figure.
Thank you for your valuable feedback. We have revised Figure 2 with the MCC tree to include posterior support on the internal nodes. We have also amended the figure legend to explain the additional coloured internal nodes. We have also amended the heatmap in Figure 2 to include additional white space between the columns to make it easier for the readers to distinguish. We didn’t change the colours in this figure as we have used the same colours throughout for the different traits reported in this study. Further, we chose to keep the AMR profiles reported in Figure 2 at the susceptible, resistant or MDR. This was done to convey the overview of the AMR profiles, and we provide detail in the AMR and HMR determinants in the Supplementary Figures and Tables.
(3) The manuscript title is not informative, as it did not study the 'dynamics' of the two genotypes. Suggest to revise the study title along the lines of main results.
Thank you for the feedback on the title. We have amended this to better reflect the main findings of the study, and it now reads as “Distinct adaptation and epidemiological success of different genotypes within Salmonella enterica serovar Dublin”
(4) The co-occurrence of AMR and heavy metal resistance genes (like mer) are quite common in Salmonella and E. coli. This is not a novel finding. The reviewer would suggest shortening the details related to heavy metal resistance in Results and Discussion, to make the writing more streamlined.
In line with the Reviewer comments, we have shortened the details in the Results and Discussion on the co-occurrence of AMR and HMR.
(5) L185: missing info after n=82.
This has been revised to now read as “n=82 from Canada”.
(6) I think Vi refers to the capsular antigen, not flagelle. Please double-check this.
Thank you for highlighting this mistake. We have revised all instances.
(7) L252-253: which statistic was used to state 'no association'. Also, there is no evidence presented to support 'no fitness cost associated with resistance and virulence."
We have removed this sentence.
(8) 320: Figure 6F is a scatterplot, not PCA. Please confirm.
The reviewer is correct, this is in fact a scatterplot. We have amended the figure legend and text.
(9) For Discussion, it would be helpful to compare the phenotype findings with that of other invasive Salmonella like Typhi or Typhimurium ST313.
Thank you for noting this, we had alluded to findings from ST313 but have now expanded include some further comparisons to S. Typhimurium ST313 and added references for these within the Discussion. The additional text now reads:
“Similar trends have been observed with S. Typhimurium ST313, which induces less inflammasome activation than ST19 during murine macrophage infection (61). This could facilitate increased replication and dissemination at later stages of infection.”
"Invasiveness of S. enterica is often linked to genome degradation (4,62–64).
However, this is mostly based on studies of human-adapted iNTS (ST313) and S. Typhi, leaving open the possibility that the additional genomic content of ST74 supports survival in diverse host species. An uncharacterised virulence factor may underlie this replication advantage.”
(10) L440: no evidence for "successful colonization" of ST74. Actually, the findings suggested otherwise.
Thank you for picking this up, we have amended the sentence to better reflect the findings. The amended text now reads as:
“It’s possible that the efficient replication of ST74 in macrophages might compensate for the absence of SPI-19, relying instead on phagocyte uptake via M cells or dendritic cells. The larger pangenome of ST74 compared to ST10 could further enhance survival within hosts.”
(11) L460-461: The data did not show an increasing trend of iNTS related to S. Dublin.
Thank you for identifying this. This sentence has been revised accordingly and now reads as:
“While the data did not indicate an increasing trend of iNTS associated with S. Dublin, the potential public health risk of this pathogen suggests it may still warrant considering it a notifiable disease, similar to typhoid and paratyphoid fever.”
(12) L465: Data were not analyzed explicitly in the context of animal vs. human. Suggest omitting 'One Health' from the conclusion.
Thank you for the suggestion. We have omitted “One Health” from the conclusion
(13) L500: Was the alignment not checked for recombination using Gubbins? The approach here is inconsistent with the method described in the subtree selected for BEAST analysis (L546).
We have now applied Gubbins to the phylogenetic tree constructed using IQTREE, and the methods and results have been updated accordingly.
(14) What was the output of Tempest? Correlation or R2 value?
We have now included the R2 value from Tempest and reported this in the manuscript.
(15) L556: marginal likelihood to allow evaluation of the best-fit model. Please rephrase to state this clearly.
We have rephrased this in the manuscript to state this clearly.
