Author response:
Reviewer #1 (Public Review):
Summary:
A nice study trying to identify the relationship between E. coli O157 from cattle and humans in Alberta, Canada.
Strengths:
(1) The combined human and animal sampling is a great foundation for this kind of study.
(2) Phylogenetic analyses seem to have been carried out in a high-quality fashion.
Weaknesses:
I think there may be a problem with the selection of the isolates for the primary analysis. This is what I'm thinking:
(1) Transmission analyses are strongly influenced by the sampling frame.
(2) While the authors have randomly selected from their isolate collections, which is fine, the collections themselves are not random.
(3) The animal isolates are likely to represent a broad swathe of diversity, because of the structured sampling of animal reservoirs undertaken (as I understand it).
(4) The human isolates are all from clinical cases. Clinical cases of the disease are likely to be closely related to other clinical cases, because of outbreaks (either detected, or undetected), and the high ascertainment rate for serious infections.
(5) Therefore, taking an equivalent number of animal and clinical isolates, will underestimate the total diversity in the clinical isolates because the sampling of the clinical isolates is less "independent" (in the statistical sense) than sampling from the animal isolates.
(6) This could lead to over-estimating of transmission from cattle to humans.
We appreciate the reviewer’s careful thoughts about our sampling strategy. We agree with points (1) and (2), and we will provide additional details on the animal collections as requested.
We agree with point (3) in theory but not in fact. As shown in Figure 3a, the cattle isolates were very closely related, despite the temporal and geographic breadth of sampling within Alberta. The median SNP distance between cattle sequences was 45 (IQR 36-56), compared to 54 (IQR 43-229) SNPs between human sequences from cases in Alberta during the same years. Additionally, as shown in Figure 2, only clade A and B isolates – clades that diverge substantially from the rest of the tree – were dominated by human cases in Alberta. We will better highlight this evidence in the revision.
We agree with the reviewer in point (4) that outbreaks can be an important confounder of phylogenetic inference. This is why we down-sampled outbreaks (based on genetic relatedness, not external designation) in our extended analyses (lines 192-194). We did not do this in the primary analysis, because there were no large clusters of identical isolates. Figure 3b shows a limited number of small clusters; however, clustered cattle isolates outnumbered clustered human isolates, suggesting that any bias would be in the opposite direction the reviewer suggests. Regarding severe cases being oversampled among the clinical isolates, this is absolutely true and a limitation of all studies utilizing public health reporting data. We will make this limitation to generalizability clearer in the discussion. However, as noted above, clinical isolates were more variable than cattle isolates, so it does not appear to have heavily biased the analysis.
We disagree with the reviewer on point (5). While the bias toward severe cases could make the human isolates less independent, the relative sampling proportions are likely to induce greater distance between clinical isolates than cattle isolates, which is exactly what we observe (see response to point (3) above). Cattle are E. coli O157:H7’s primary reservoir, and humans are incidental hosts not able to sustain infection chains long-term. Not only is the bacteria prevalent among cattle, cattle are also highly prevalent in Alberta. Thus, even with 89 sampling points, we are still capturing a small proportion of the E. coli O157:H7 in the province. Being able to sample only a small proportion of cattle’s E. coli O157:H7 increases the likelihood of only sampling from the center of the distribution, making extreme cases such as that shown at the very bottom of the tree in Figure 3b, rare and important. In comparison, sampling from human cases constitutes a higher proportion of human infections relative to cattle, and is therefore more representative of the underlying distribution, including extremes. We will add this point to the limitations. As with the clustering above, if anything, this outcome would have biased the study away from identifying cattle as the primary reservoir. Additionally, the relatively small proportion of cattle sampled makes our finding that 15.7% of clinical isolates were within 5 SNPs of a cattle isolate, the distance most commonly used to indicate transmission for E. coli O157:H7, all the more remarkable.
Because of the aforementioned points, we disagree with the reviewer’s conclusion in point (6). We believe transmission from cattle-to-humans is likely underestimated for the reasons given above. Not only do all prior studies indicate ruminants as the primary reservoirs of E. coli O157:H7, and humans as only incidental hosts, our specific data do not support the reviewer’s individual contentions. That said, we will conduct a sensitivity analysis as recommended to determine the impact of sampling and inclusion of the small clusters on our primary findings.
(7) We hypothesize that the large proportion of disease associated with local transmission systems is a principal cause of Alberta's high E. coli O157:H7 incidence" - this seems a bit tautological. There is a lot of O157 because there's a lot of transmission. What part of the fact it is local means that it is a principal cause of high incidence? It seems that they've observed a high rate of local transmission, but the reasons for this are not apparent, and hence the cause of Alberta's incidence is not apparent. Would a better conclusion not be that "X% of STEC in Alberta is the result of transmission of local variants"? And then, this poses a question for future epi studies of what the transmission pathway is.
The reviewer is correct, and the suggestion for the direction of future studies was our intent with this statement. We will revise it.
Reviewer #2 (Public Review):
This study identified multiple locally evolving lineages transmitted between cattle and humans persistently associated with E. coli O157:H7 illnesses for up to 13 years. Furthermore, this study mentions a dramatic shift in the local persistent lineages toward strains with the more virulent stx2a-only profile. The authors hypothesized that this phenomenon is the large proportion of disease associated with local transmission systems is a principal cause of Alberta's high E. coli O157:H7 incidence. These opinions more effectively explain the role of the cattle reservoir in the dynamics of E. coli O157:H7 human infections.
(1) The authors acknowledge the possibility of intermediate hosts or environmental reservoirs playing a role in transmission. Further discussion on the potential roles of other animal species commonly found in Alberta (e.g., sheep, goats, swine) could enhance the understanding of the transmission dynamics. Were isolates from these species available for analysis? If not, the authors should clearly state this limitation.
We will expand the discussion of other species in Alberta, as suggested, including other livestock, wildlife, and the potential role of birds and flies. Unfortunately, we did not have sequences available from other species, and we will add this to the limitations. Sequences from other species may be available from sequences collected by others, which as we note in the limitations do not have sufficient metadata to assign them to Alberta vs. the rest of Canada. While we have requested this data, we have been unsuccessful in obtaining it. We will continue to pursue it.
(2) The focus on E. coli O157:H7 is understandable given its prominence in Alberta and the availability of historical data. However, a brief discussion on the potential applicability of the findings to non-O157 STEC serogroups, and the limitations therein, would be beneficial. Are there reasons to believe the transmission dynamics would be similar or different for other serogroups?
We appreciate this comment and will expand our discussion of relevance to non-O157 STEC. Other authors have proposed that transmission dynamics differ, and studies of STEC risk factors, including our own, support this. However, there has been very little direct study of non-O157 transmission dynamics and there is even less cross-species genomic and metadata available for non-O157 isolates of concern.
(3) The authors briefly mention the need for elucidating local transmission systems to inform management strategies. A more detailed discussion on specific public health interventions that could be targeted at the identified LPLs and their potential reservoirs would strengthen the paper's impact.
We agree with the reviewer that this would be a good addition to the manuscript. The public health implications for control are several and extend to non-STEC reportable zoonotic enteric infections, such as Campylobacter and Salmonella. We will add a discussion of these.
(4) Understanding the relationship between specific risk factors and E. coli O157:H7 infections is essential for developing effective prevention strategies. Have case-control or cohort studies been conducted to assess the correlation between identified risk factors and the incidence of E. coli O157:H7 infections? What methodologies were employed to control for potential confounders in these studies?
Yes, there have been several case-control studies of reported cases. Many of these are referenced in the discussion in terms of the contribution of different sources to infection. However, we will add a more explicit discussion of risk factors.
(5) The study's findings are noteworthy, particularly in the context of E. coli O157:H7 epidemiology. However, the extent to which these results can be replicated across different temporal and geographical settings remains an open question. It would be constructive for the authors to provide additional data that demonstrate the replication of their sampling and sequencing experiments under varied conditions. This would address concerns regarding the specificity of the observed patterns to the initial study's parameters.
We appreciate the reviewer’s comment, as we are currently building on this analysis with an American dataset with different types of data available than were used in this study. We will add a discussion of this. We will also be adding a sensitivity analysis to the manuscript simulating a different sampling approach, which should also be informative to this question.
