Enterobacterales plasmid sharing amongst human bloodstream infections, livestock, wastewater, and waterway niches in Oxfordshire, UK
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Curated by eLife
eLife assessment
This important study presents valuable findings on the dissemination of plasmids. In an analysis of five major Enterobacterales genera, the authors convincingly demonstrate that similar plasmids are shared between genera, species, and clones, both within and between ecological niches. Given the size of the dataset and the very detailed level of analysis this study importantly contributes to insights into to the flow of plasmids, including those carrying antimicrobial resistance genes, across niches.
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Abstract
Plasmids enable the dissemination of antimicrobial resistance (AMR) in common Enterobacterales pathogens, representing a major public health challenge. However, the extent of plasmid sharing and evolution between Enterobacterales causing human infections and other niches remains unclear, including the emergence of resistance plasmids. Dense, unselected sampling is essential to developing our understanding of plasmid epidemiology and designing appropriate interventions to limit the emergence and dissemination of plasmid-associated AMR. We established a geographically and temporally restricted collection of human bloodstream infection (BSI)-associated, livestock-associated (cattle, pig, poultry, and sheep faeces, farm soils) and wastewater treatment work (WwTW)-associated (influent, effluent, waterways upstream/downstream of effluent outlets) Enterobacterales. Isolates were collected between 2008 and 2020 from sites <60 km apart in Oxfordshire, UK. Pangenome analysis of plasmid clusters revealed shared ‘backbones’, with phylogenies suggesting an intertwined ecology where well-conserved plasmid backbones carry diverse accessory functions, including AMR genes. Many plasmid ‘backbones’ were seen across species and niches, raising the possibility that plasmid movement between these followed by rapid accessory gene change could be relatively common. Overall, the signature of identical plasmid sharing is likely to be a highly transient one, implying that plasmid movement might be occurring at greater rates than previously estimated, raising a challenge for future genomic One Health studies.
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Author Response
Reviewer #2 (Public Review):
In their study the authors aimed to investigate the dissemination of Enterobacterales plasmids between geographically and temporally restricted isolates recovered from different niches, such as human blood stream infections, livestock, and wastewater treatment works. By using a very strict similarity threshold (Mash distance < 0.0001) the authors identified so-called groups of near-identical plasmids in which plasmids from different genera, species, and clonal background co-clustered. Also, 8% of these groups contained plasmids from different niches (e.g., human BSI and livestock) while in 35% of these cross-niche groups plasmids carried antimicrobial resistance (AMR) genes suggesting recent transfer of AMR plasmids between these ecological niches.
Next, the authors set-out to examine …
Author Response
Reviewer #2 (Public Review):
In their study the authors aimed to investigate the dissemination of Enterobacterales plasmids between geographically and temporally restricted isolates recovered from different niches, such as human blood stream infections, livestock, and wastewater treatment works. By using a very strict similarity threshold (Mash distance < 0.0001) the authors identified so-called groups of near-identical plasmids in which plasmids from different genera, species, and clonal background co-clustered. Also, 8% of these groups contained plasmids from different niches (e.g., human BSI and livestock) while in 35% of these cross-niche groups plasmids carried antimicrobial resistance (AMR) genes suggesting recent transfer of AMR plasmids between these ecological niches.
Next, the authors set-out to examine the wider plasmid population structure by clustering plasmids based on 21-mer distributions capturing both coding and non-coding plasmid regions and using a data-driven threshold to build plasmid networks and the Louvain algorithm to detect the plasmid clusters. This yielded 247 clusters of which almost half of the clusters contained BSI plasmids and plasmids from at least one other niche, while 21% contained plasmids carrying AMR genes. To further assess cross-niche plasmids similarities, the authors performed an additional plasmid pangenome-like analysis. This highlighted patterns of gain and loss of accessory plasmid functions in the background of a conserved plasmid backbone.
By comparing plasmid core gene or plasmid backbone phylogenies with chromosome core gene phylogenies, the authors assessed in more detail the dissemination of plasmids between humans and livestock. This indicated that, at least for E. coli, AMR dissemination between human and livestock-associated niches is most likely not the result of clonal spread but that plasmid movement plays an important role in cross-niche dissemination of AMR.
Based on these data the authors conclude that in Enterobacterales plasmid spread between different ecological niches could be relatively common, even might be occurring at greater rates than estimated, as signatures of near-identity could be transient once plasmids occupy and adept to a different niche. After such a host jump, subsequent acquisition, and loss of parts of the accessory plasmid gene content, as a result of plasmid evolution after inter-host transfer, may obscure this near-identity signature. As stated by the authors, this will raise challenges for future One Health-based genomic studies.
Strengths
The article is well written with a clear structure. The authors have used for their analysis a comprehensive collection of more than 1500 whole genome sequenced and fully assembled isolates, yielding a dataset of more than 3600 fully assembled plasmids across different bacterial genera, species, clonal backgrounds, and ecological niches. A strong asset of the collection, especially when analyzing dissemination of AMR contained on plasmids, is that isolates were geographically and temporally restricted. Bioinformatic analyses used to discern plasmid similarity are beyond state-of-the-art. The conclusions about dissemination of plasmids between genera, species, clonal background and across ecological niches are well supported by the data. Although conclusions about inter-host plasmid dissemination patterns may have been drawn before, this is to my knowledge the first time that patterns of dissemination of plasmids have been studied at such a high-level of detail in such a well selected dataset using so many fully assembled genomes.
Weaknesses
One conclusion that is not entirely supported by the data is the general statement in the discussion that "cross-niche plasmid in not driven by clonal lineages". From the tanglegram, displaying the low congruence between the plasmid and chromosome core gene phylogeny in E. coli, this conclusion is probably valid for E. coli, but this not necessarily means that this is also the case for the other Enterobacterales genera and species included in this study. For these other genera, the data supporting this conclusion are not given, probably because total number of isolates for certain genera were low, or because certain niches were clearly underrepresented in certain genera.
Thank you for reviewing our manuscript.
We agree that this statement in the conclusion was too general, and have adapted it (lines 407-409):
“By examining plasmid relatedness compared to bacterial host relatedness in E. coli, we demonstrated that plasmids seen across different niches are not necessarily associated with clonal lineages”
In the limitations section of the Discussion, we have also referenced this specifically as a limitation (lines 422-424):
“Although we evaluated four bacterial genera, 72% (1,044/1,458) of our sequenced isolates were E. coli, and so our analyses and findings are particularly focused on this species.”
Furthermore, the BSI as well as the livestock niches were analyzed as single niches while the BSI niche included both nosocomial and community-derived BSI isolates and the Livestock niche included samples from different livestock-related hosts. Given the fact that a substantial number of plasmids were available from cattle, sheep, pigs, and poultry, it would be interesting to see whether particular livestock hosts were more frequently found in the cross-niche plasmid clusters than other livestock hosts and whether the BSI plasmids in these cross-niche clusters were predominantly of community or nosocomial origin.
We agree that analyses which distinguish between nosocomial/community acquired BSI isolates would be interesting further work, but are beyond the scope of this study. Our analysis of the BSI/livestock cross-niche near-identical plasmid groups details the livestock hosts involved (lines 144-154). Briefly, of the n=8 BSI/livestock cross-niche groups, these involved
• pig/poultry (1/8)
• poultry (1/8)
• pig (2/8)
• sheep (3/8)
• cattle/pig/poultry (1/8)
We have added a note of explanation in the methods to explain how the distance threshold we use for near-identical clustering is maximally conservative at small plasmid sizes (a single SNP produces a new plasmid cluster) but remains highly conservative (tens of SNPs) at large plasmid sizes.
We have carefully considered the point about whether particular hosts were more frequently found in cross-niche plasmid clusters. However, we do not think it is easy to infer whether a particular livestock host is represented more frequently in these cross-niche events than would be expected from chance, given the low density of the sampling.
We have reorganised the paragraph in lines 144-154 to provide more clarity on the groups’ niches.
“Sharing between BSI and livestock-associated isolates was supported by 8/17 cross-niche groups (n=45 plasmids). Of these, n=3/8 groups contained BSI/sheep plasmids: one group contained mobilisable Col-type plasmids, the remaining two groups contained conjugative FIB-type plasmids. Of these, one group contained plasmids carrying the AMR genes aph(3'')-Ib, aph(6)-Id, blaTEM-1, dfrA5, sul2, and the other group contained plasmids carrying the MDR efflux pump protein robA (see Materials and Methods). A further n=2/8 groups contained BSI/pig mobilisable Col-type plasmids, of which one group other carried the AMR genes aph(3'')-Ib, aph(6)-Id, dfrA14, and sul2. Lastly, n=1/8 groups contained BSI/poultry non-mobilisable Col-type plasmids, n=1/8 contained BSI/pig/poultry/influent non-mobilisable Col-type plasmids, and n=1/8 contained BSI/cattle/pig/poultry/influent mobilisable Col-type plasmids.”
We have also added this as a limitation in the discussion (lines 424-426):
“Additionally, we did not sample livestock-associated niches densely enough to explore individual livestock types (cattle/pigs/poultry/sheep) sharing plasmids with BSI isolates (see Appendix 1 Fig. 9).”
We have already recognised that our culture methods may have affected our sensitivity to detect Klebsiella spp. isolates in the livestock/environmental samples – we have expanded on this to explicitly highlight that this may have affected our capacity to evaluate Klebsiella-associated plasmids (lines 443-444):
“This limited our ability to study the epidemiology of livestock Klebsiella plasmids.”
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eLife assessment
This important study presents valuable findings on the dissemination of plasmids. In an analysis of five major Enterobacterales genera, the authors convincingly demonstrate that similar plasmids are shared between genera, species, and clones, both within and between ecological niches. Given the size of the dataset and the very detailed level of analysis this study importantly contributes to insights into to the flow of plasmids, including those carrying antimicrobial resistance genes, across niches.
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Reviewer #1 (Public Review):
In this study 1458 Enterobacterales isolates, derived from animals, waste-water and human bloodstream infections, were genetically characterized. This also yielded 3697 plasmids and many AMR genes.
All isolates were derived in a restricted geographical region and within a few years time. They defined "groups of near-identical plasmids" with plasmids derived from different genera, species, and clonal background; 8% of these groups contained plasmids from the different ecological niches and 35% of these cross-niche groups plasmids carried AMR genes. This fits with the concept of recent transfer of AMR plasmids between these ecological niches. Through detailed analyses they provide evidence that for E. coli, AMR dissemination between human and livestock-associated niches is most likely not the result of clonal …
Reviewer #1 (Public Review):
In this study 1458 Enterobacterales isolates, derived from animals, waste-water and human bloodstream infections, were genetically characterized. This also yielded 3697 plasmids and many AMR genes.
All isolates were derived in a restricted geographical region and within a few years time. They defined "groups of near-identical plasmids" with plasmids derived from different genera, species, and clonal background; 8% of these groups contained plasmids from the different ecological niches and 35% of these cross-niche groups plasmids carried AMR genes. This fits with the concept of recent transfer of AMR plasmids between these ecological niches. Through detailed analyses they provide evidence that for E. coli, AMR dissemination between human and livestock-associated niches is most likely not the result of clonal spread but rather that plasmids transit between ecological niches.
Strengths
This is - to the best of my knowledge - one of the largest and most detailed studies elucidating the epidemiology of plasmids and AMR genes in different ecological niches.
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Reviewer #2 (Public Review):
In their study the authors aimed to investigate the dissemination of Enterobacterales plasmids between geographically and temporally restricted isolates recovered from different niches, such as human blood stream infections, livestock, and wastewater treatment works. By using a very strict similarity threshold (Mash distance < 0.0001) the authors identified so-called groups of near-identical plasmids in which plasmids from different genera, species, and clonal background co-clustered. Also, 8% of these groups contained plasmids from different niches (e.g., human BSI and livestock) while in 35% of these cross-niche groups plasmids carried antimicrobial resistance (AMR) genes suggesting recent transfer of AMR plasmids between these ecological niches.
Next, the authors set-out to examine the wider plasmid …
Reviewer #2 (Public Review):
In their study the authors aimed to investigate the dissemination of Enterobacterales plasmids between geographically and temporally restricted isolates recovered from different niches, such as human blood stream infections, livestock, and wastewater treatment works. By using a very strict similarity threshold (Mash distance < 0.0001) the authors identified so-called groups of near-identical plasmids in which plasmids from different genera, species, and clonal background co-clustered. Also, 8% of these groups contained plasmids from different niches (e.g., human BSI and livestock) while in 35% of these cross-niche groups plasmids carried antimicrobial resistance (AMR) genes suggesting recent transfer of AMR plasmids between these ecological niches.
Next, the authors set-out to examine the wider plasmid population structure by clustering plasmids based on 21-mer distributions capturing both coding and non-coding plasmid regions and using a data-driven threshold to build plasmid networks and the Louvain algorithm to detect the plasmid clusters. This yielded 247 clusters of which almost half of the clusters contained BSI plasmids and plasmids from at least one other niche, while 21% contained plasmids carrying AMR genes. To further assess cross-niche plasmids similarities, the authors performed an additional plasmid pangenome-like analysis. This highlighted patterns of gain and loss of accessory plasmid functions in the background of a conserved plasmid backbone.
By comparing plasmid core gene or plasmid backbone phylogenies with chromosome core gene phylogenies, the authors assessed in more detail the dissemination of plasmids between humans and livestock. This indicated that, at least for E. coli, AMR dissemination between human and livestock-associated niches is most likely not the result of clonal spread but that plasmid movement plays an important role in cross-niche dissemination of AMR.
Based on these data the authors conclude that in Enterobacterales plasmid spread between different ecological niches could be relatively common, even might be occurring at greater rates than estimated, as signatures of near-identity could be transient once plasmids occupy and adept to a different niche. After such a host jump, subsequent acquisition, and loss of parts of the accessory plasmid gene content, as a result of plasmid evolution after inter-host transfer, may obscure this near-identity signature. As stated by the authors, this will raise challenges for future One Health-based genomic studies.
Strengths
The article is well written with a clear structure. The authors have used for their analysis a comprehensive collection of more than 1500 whole genome sequenced and fully assembled isolates, yielding a dataset of more than 3600 fully assembled plasmids across different bacterial genera, species, clonal backgrounds, and ecological niches. A strong asset of the collection, especially when analyzing dissemination of AMR contained on plasmids, is that isolates were geographically and temporally restricted. Bioinformatic analyses used to discern plasmid similarity are beyond state-of-the-art. The conclusions about dissemination of plasmids between genera, species, clonal background and across ecological niches are well supported by the data. Although conclusions about inter-host plasmid dissemination patterns may have been drawn before, this is to my knowledge the first time that patterns of dissemination of plasmids have been studied at such a high-level of detail in such a well selected dataset using so many fully assembled genomes.Weaknesses
One conclusion that is not entirely supported by the data is the general statement in the discussion that "cross-niche plasmid in not driven by clonal lineages". From the tanglegram, displaying the low congruence between the plasmid and chromosome core gene phylogeny in E. coli, this conclusion is probably valid for E. coli, but this not necessarily means that this is also the case for the other Enterobacterales genera and species included in this study. For these other genera, the data supporting this conclusion are not given, probably because total number of isolates for certain genera were low, or because certain niches were clearly underrepresented in certain genera.Furthermore, the BSI as well as the livestock niches were analyzed as single niches while the BSI niche included both nosocomial and community-derived BSI isolates and the Livestock niche included samples from different livestock-related hosts. Given the fact that a substantial number of plasmids were available from cattle, sheep, pigs, and poultry, it would be interesting to see whether particular livestock hosts were more frequently found in the cross-niche plasmid clusters than other livestock hosts and whether the BSI plasmids in these cross-niche clusters were predominantly of community or nosocomial origin.
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