Pathogen invasion-dependent tissue reservoirs and plasmid-encoded antibiotic degradation boost plasmid spread in the gut
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Evaluation Summary:
This work describes an important feature of within-host acquisition of antibiotic resistance. This is a follow-up to their recent publication (Bakkeren et al. 2019), and complements their finding of persister cells in the tissues, to show that also chronic, tissue residing bacteria can provide plasmid tissue reservoirs. The experiments the authors performed are elegant and timely. This manuscript will be of interest to readers in the fields of infection biology, plasmid ecology, gut microbiomes, and antimicrobial resistance.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Many plasmids encode antibiotic resistance genes. Through conjugation, plasmids can be rapidly disseminated. Previous work identified gut luminal donor/recipient blooms and tissue-lodged plasmid-bearing persister cells of the enteric pathogen Salmonella enterica serovar Typhimurium ( S .Tm) that survive antibiotic therapy in host tissues, as factors promoting plasmid dissemination among Enterobacteriaceae. However, the buildup of tissue reservoirs and their contribution to plasmid spread await experimental demonstration. Here, we asked if re-seeding-plasmid acquisition-invasion cycles by S .Tm could serve to diversify tissue-lodged plasmid reservoirs, and thereby promote plasmid spread. Starting with intraperitoneal mouse infections, we demonstrate that S .Tm cells re-seeding the gut lumen initiate clonal expansion. Extended spectrum beta-lactamase (ESBL) plasmid-encoded gut luminal antibiotic degradation by donors can foster recipient survival under beta-lactam antibiotic treatment, enhancing transconjugant formation upon re-seeding. S .Tm transconjugants can subsequently re-enter host tissues introducing the new plasmid into the tissue-lodged reservoir. Population dynamics analyses pinpoint recipient migration into the gut lumen as rate-limiting for plasmid transfer dynamics in our model. Priority effects may be a limiting factor for reservoir formation in host tissues. Overall, our proof-of-principle data indicates that luminal antibiotic degradation and shuttling between the gut lumen and tissue-resident reservoirs can promote the accumulation and spread of plasmids within a host over time.
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Evaluation Summary:
This work describes an important feature of within-host acquisition of antibiotic resistance. This is a follow-up to their recent publication (Bakkeren et al. 2019), and complements their finding of persister cells in the tissues, to show that also chronic, tissue residing bacteria can provide plasmid tissue reservoirs. The experiments the authors performed are elegant and timely. This manuscript will be of interest to readers in the fields of infection biology, plasmid ecology, gut microbiomes, and antimicrobial resistance.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
In this work, Bakkeren et al study the ability of tissue lodged Salmonella to reseed the gut, initiate clonal expansion and serve as recipients for transconjugation of antibiotic resistance plasmids by gut luminal donor bacteria. The authors use i.p injection of Salmonella to induce tissue reservoir recipient cells. They then treat with streptomycin to clear the microbiota and introduce donors orally (either e.coli or Salmonella). The authors show that in this model, tissue resident recipients migrate to the gut, and produce transconjugates that then reseed the tissue. Using tagged strains and modelling, the authors suggest that recipient migration from the tissue is a rate-limiting step. The authors further confirm that these tissue reservoirs can serve for plasmid transfer in future reinfections of the …
Reviewer #1 (Public Review):
In this work, Bakkeren et al study the ability of tissue lodged Salmonella to reseed the gut, initiate clonal expansion and serve as recipients for transconjugation of antibiotic resistance plasmids by gut luminal donor bacteria. The authors use i.p injection of Salmonella to induce tissue reservoir recipient cells. They then treat with streptomycin to clear the microbiota and introduce donors orally (either e.coli or Salmonella). The authors show that in this model, tissue resident recipients migrate to the gut, and produce transconjugates that then reseed the tissue. Using tagged strains and modelling, the authors suggest that recipient migration from the tissue is a rate-limiting step. The authors further confirm that these tissue reservoirs can serve for plasmid transfer in future reinfections of the gut. Finally, the authors suggest that vaccines and gut luminal e.coli harboring beta lactam resistance plasmids can protect tissue resident recipients for further transconjugation.
This work describes an important feature of within-host acquisition of antibiotic resistance. This is a follow-up to their recent publication (Bakkeren et al 2019), and complements their finding of persister cells in the tissues, to show that also chronic, tissue residing bacteria can provide plasmid tissue reservoirs. The experiments the authors performed are elegant and timely. However, their conclusions require further experimental evidence. Importantly, the authors should provide experimental support in a chronic model of infection. Furthermore, insights into the mechanism of tissue reseeding and possible effects on the intact microbiota are required.
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Reviewer #2 (Public Review):
In the study "Pathogen invasion-dependent tissue reservoirs and plasmid-encoded antibiotic degradation boost plasmid spread in the gut", Bakkeren, Hardt and colleagues investigate the dynamics of antibiotic resistance-encoding plasmid acquisition in Salmonella Typhimurium, using mouse models of persistent Salmonella infection and antibiotic persistent Salmonella infection. Specifically, the authors initially show that tissue-resident Salmonella can re-seed the gut lumen, acquire antibiotic resistance plasmids through trans-conjugation, and subsequently re-invade the mesenteric lymph nodes following trans-conjugation. They go on to show that trans-conjugation can occur if the donor is either Salmonella or E. coli, and that trans-conjugation can occur both in the absence of antibiotic treatment …
Reviewer #2 (Public Review):
In the study "Pathogen invasion-dependent tissue reservoirs and plasmid-encoded antibiotic degradation boost plasmid spread in the gut", Bakkeren, Hardt and colleagues investigate the dynamics of antibiotic resistance-encoding plasmid acquisition in Salmonella Typhimurium, using mouse models of persistent Salmonella infection and antibiotic persistent Salmonella infection. Specifically, the authors initially show that tissue-resident Salmonella can re-seed the gut lumen, acquire antibiotic resistance plasmids through trans-conjugation, and subsequently re-invade the mesenteric lymph nodes following trans-conjugation. They go on to show that trans-conjugation can occur if the donor is either Salmonella or E. coli, and that trans-conjugation can occur both in the absence of antibiotic treatment (persistent/chronic infection model) or following antibiotic treatment (antibiotic persistent Salmonella infection model). The authors show that re-seeding of the gut lumen by recipient tissue-resident Salmonella is a rare event that is the rate-limiting step for trans-conjugant formation. They also show that, following trans-conjugation, recipient Salmonella can occasionally re-invade the mesenteric lymph nodes (MLNs). Using a mathematical model based on their experimental data, the authors predict that trans-conjugant formation and re-invasion of MLNs from the gut lumen are dependent on the carrying capacity of the recipient population in the gut, implying that colonisation resistance is likely an important factor in limiting plasmid spread dynamics. The authors go on to show that newly formed transconjugants that have re-invaded MLNs can act as both donors and recipients in further trans-conjugation events following antibiotic treatment and re-seeding of the gut lumen. This highlights that re-seeding of the gut lumen, trans-conjugation, and re-invasion of host tissue can occur cyclically. Finally, the authors show that ESBL resistance plasmids from luminal donors facilitate the survival and expansion of tissue-resident Salmonella recipients that re-seed the gut lumen during treatment with beta-lactam antibiotics, and that, in turn, this enables trans-conjugation of ESBL resistance plasmids to beta-lactam-susceptible recipient Salmonella during beta-lactam treatment.
The study is well done and focusses on a very timely and important subject (the acquisition and spread of antibiotic resistance plasmids) using an appropriate model system. I note however that this work is largely a validation and further expansion of the work this group has recently published (Bakkeren et al., Nature 2019; PMID: 31485077). The primary claims of the paper are supported by the data presented, with the following exception: I am not fully convinced that the authors have clearly shown that the second trans-conjugation event (described in Figure 3B, S7A-B) is necessarily between the initial trans-conjugant and the second recipient. Although unlikely, it does remain formally possible that the initial E coli donor acted as donor in this second trans-conjugation event.
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Reviewer #3 (Public Review):
Previously, Bakkeren and colleagues (Nature, 2019) showed that 'persister' cells of Salmonella Typhimurium, that survive antibiotic treatment in host tissues, could act as a source of antibiotic resistance plasmids when they re-seed the gut. Here, the authors expand on this work to show that tissue-resident Salmonella can also act as a plasmid recipient when it re-seeds the gut, and can then essentially capture gut-resident plasmids into long-term storage when it re-invades tissues. These experiments are performed by sequentially infecting mice with different combinations of plasmid-free and plasmid-containing Salmonella and E. coli and looking for the emergence of transconjugants in different tissues over time. Using these data, the authors parameterise a mathematical model of infection, enabling them to …
Reviewer #3 (Public Review):
Previously, Bakkeren and colleagues (Nature, 2019) showed that 'persister' cells of Salmonella Typhimurium, that survive antibiotic treatment in host tissues, could act as a source of antibiotic resistance plasmids when they re-seed the gut. Here, the authors expand on this work to show that tissue-resident Salmonella can also act as a plasmid recipient when it re-seeds the gut, and can then essentially capture gut-resident plasmids into long-term storage when it re-invades tissues. These experiments are performed by sequentially infecting mice with different combinations of plasmid-free and plasmid-containing Salmonella and E. coli and looking for the emergence of transconjugants in different tissues over time. Using these data, the authors parameterise a mathematical model of infection, enabling them to infer likely rates of conjugation and bacterial migration between gut and tissues.
The concept is intriguing and extends our understanding of plasmid ecology and infection biology. The ability of plasmids to become acquired from the gut to the tissue reservoir has interesting implications for the maintenance and dissemination of plasmid-borne genes in microbial communities.
This work meaningfully extends the previous study, which showed that plasmids could emerge from persistent reservoirs into the gut, to show that plasmids can be captured from the gut into persistent reservoirs. The experiments are well-designed and generally suitable for testing the specific questions under consideration. The use of mathmatical models enables enhanced insight to be gained from the experimental data. In general, the data are clearly presented and interpreted.
The main scientific issue I have with the manuscript is that the experimental setup presented by the authors is very artificial, involving intraperitoneal inoculation of gut pathogens, antibiotic clearance of resident gut microbiota, and strong selection for plasmid carriage. The authors convincingly justify their choices in the manuscript, and I think that for a proof-of-principle study their decisions are appropriate. Still, I feel that if tissue 'storage' of plasmids indeed plays an important role in epidemiology and evolution, some predictions could be made about diversity of mobile genetic elements in tissues vs. gut lumen under natural conditions (for example), which, if not testable with existing genomic and metagenomic data within the scope of the current work, is an important subject of investigation in future studies.
A further issue with the authors' conceptual model that requires some attention: for plasmids to be 'stored' as 'a record' in tissues, it suggests that plasmids and bacteria do not change significantly in tissues over time. What evidence is there for tissue-lodged bacteria to undergo conjugation, or lose plasmids, or become displaced by newly-incoming populations? If plasmids can conjugate or displace one another in tissues, the role for reseeding into the gut becomes less dominant. There may also be implications for the mathematical model, if tissues can act as a source of transconjugants as well as recipients.
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