1. Author Response:

    Reviewer #1 (Public Review):

    The study by Diebold et al. describes a fast and scalable method that allows to link bacterial plasmids to the organisms that harbor them. The authors then go on to apply this technique to track horizontal gene transfer in an complex bacterial population originating from clinical samples. There is no doubt that the development of such methodologies for better tracking plasmidic resistance genes and following horizontal gene transfer events is very important. The authors do a good job in optimizing their method to be a one step process that has high sensitivity and relatively low error, while it can also be scaled, automated and used with multiplex primers. Subsequently, they apply this method to two clinical patient samples for which metagenomic data is available. In this case, they correctly identify expected relationships between beta-lactamase genes and specific bacterial taxa (and in particular K. pneumoniae), but also find that the same beta-lactamase genes are associated with organisms of the microbiome. With the exception of providing evidence that the association of particular genes with multiple organisms is not due to physical association of the bacteria in question, this is an interesting study putting forward a much needed technique for the study of antibiotic resistance but also other relationships in complex bacterial mixtures.

    We are very thankful for the positive review and the reviewer’s suggestion that we distinguish between gene transfer and physical association. We provide a detailed response to this in major point #1 of the review summary, but to summarize, we performed an OIL-PCR experiment to confirm that the results are indeed due to physical association of the bacteria and updated our manuscript accordingly.

    Reviewer #2 (Public Review):

    Diebold et al. developed a simplified and improved version of the epicPCR method applied to environmental samples. The results section describes well how they perform their development and support the easy to use application. They clearly demonstrate that their methods could be used to screen association of specific genes to taxonomic markers in environmental microbial populations. They then apply their methods on human gut samples ranging from hospitalized patients and demonstrate demonstrate the utility of their methods to characterize the hosts of different targeted genes (notably AMR and plasmid related genes). However, most of their results are based on previous studies on the same sample. Therefore, it appears difficult to know how their method can be used on new samples. Do they need to redo a classical metagenomic analysis in order to obtain data on new samples ? What kind of metagenomic analysis is mandatory before performing their methods ? What is the depth of the metagenomic analysis ? Those are important questions as it will be clearly more expensive to perform the whole metagenomic analysis.

    Thank you for pointing out the need to explain possible screening methods for OIL-PCR on unsequenced samples. We chose to use sequenced stool samples for testing the method in order to provide parallel validation of our results; however, we agree that metagenomic sequencing is not a practical or cost-effective way to select samples for OIL-PCR. qPCR is a more practical method to pre-screen samples for target genes before performing OIL, but we failed to include this important point in our discussion.

    Since drafting and submitting the manuscript, we have demonstrated that the three primers designed for OIL (forward, fusion, and nested primers) can easily be converted into probe- based qPCR assays by designing a fluorescent probe with the nested primer sequence. We have updated the discussion to convey this important feature of OIL-PCR.

    The conclusion of the paper is well supported by data but the overall approach on new sample is never discussed. Moreover, the title appear somehow misleading as their methods do not allow to clearly identify plasmids but rather to link some targeted genes to taxonomic markers.

    Reviewer #3 (Public Review):

    This manuscript is composed of two parts. The first part describes development of an emulsion-based PCR fusion method, called OIL-PCR, for matching two specific gene sequences from the same cell. In this report these are beta-lactamase genes from the V4 section of rRNA, allowing the matching of this horizontally transferred gene with its donor sequence. The second part is a demonstration project that features the use of OIL-PCR to monitor horizontal transfer of beta-lactam genes between gut bacteria from the metagenomes of two neutropenic patients. OIL-PCR was set to multiplexed class A beta-lactam genes. This is a descriptive study that largely recapitulates a previously published work on these samples showing that the relatively unstudied Romboutsia commensal genus is a carrier of these plasmid-borne genes in patient metagenomes.

    Overall, this is a well-written manuscript. Data were comprehensively analyzed with appropriate controls. The figures are excellent.

    OIL-PCR is a derived of other fusion PCR methods, especially epicPCR. There are some nice technical improvements described here, e.g efficient lysis within emulsion droplets using Ready-Lyse lysozyme. This is an incremental technical advance for a fairly niche application (where you have known target genes and are concerned about potential culture-bias) but it may be useful in particular for understanding HGT in microbiomes. There are some problems with the method that are brought to the foreground by the authors rather than quietly dropped, which is commendable.

    Thank you for acknowledging our effort to be up front about the strengths and weaknesses of OIL-PCR. We hope that this information will help inform other researchers in applying this method.

    One problem appears to be that the necessary dilution for single-cell PCR reduces the taxonomic diversity of the metagenome. The only way around this to perform efficient sampling appears to be to perform multiple independent sequencing experiments and pool the results. Another feature of the system is that the accuracy falls slightly as the proportion of the target sequence in the community increases for reasons that are not discussed. However, this effect is not great (97% accuracy at 10% proportion) and most applications, the target cells will be a much lower proportion of the community.

    The results of the demonstration study on metagenomes from neutropenic patients are clearly described and provide a nicely worked example of combining this directed method with metagenome sequencing. The significance is limited but gives some descriptive hits about the mechanism of HGT between Romboutsia and Klebsiella.

    Other points:

    Unfortunately, there was no comparative test where the same samples were run against "competing" technologies (e.g sequencing of cultured beta-lactam resistant strains, epicPCR, Hi-C or single-cell) to directly compare strengths (and weaknesses) of OIL-PCR.

    Thank you for this fair criticism that we did not compare OIL-PCR to other available methods. We address comparing OIL-PCR to Hi-C in our response to major point #4 (above). With regards to epicPCR, we did consider comparing OIL-PCR to epicPCR, but decided against it for two main reasons: 1) Acquiring all the reagents necessary to perform epicPCR was cost- prohibitive (over $1,000 for the one demonstration experiment), and 2) because a large motivation for the development of OIL-PCR is the difficulty of performing epicPCR. Although we believe that both epicPCR and OIL-PCR are robust methods, OIL-PCR is a shorter protocol that does not rely on hazardous, costly and difficult to obtain reagents. We were concerned an inexperienced attempt by us to perform epicPCR would likely have yielded poor results and would not provide a fair comparison. Overall, we feel that the validation experiments we perform with OIL-PCR are enough to highlight both the strengths and weakness of the method.

    As protocol development is central to this manuscript paper, and one of the main advantages claimed for OIL-PCR is ease of use, the supplement should contain a detailed protocol for control sample with a list of equipment and reagents needed and what results should be obtained. This could easily be adapted from the methods section, which is highly detailed. What is the estimated cost-per sample of this procedure and how does it compare roughly with other methods, - EPIC-PCR and culture-based?

    Thank you for the suggestion that we provide a detailed protocol. We hope that the inclusion of this step-by-step protocol will enable more labs to adopt the method. The cost of OIL is approximately $15 per replicate. The cost is largely driven by the large amount of Phusion polymerase needed, which is the same as in epicPCR. Culturing may be less expensive depending on the cost of reagents needed for media, antibiotics etc, but we do not feel the two are comparable. For example, even though we show that Romboutsia did not acquire resistance genes in this case, even if it had, culturing would not have captured it due to the difficult and specific culturing conditions required for growing most Romboutsia strains.

    Line 197-198 reference needed to the Kent et al study here? What is the reason that the Hi-C results from this manuscript are not compared to the results of the OIL-PCR experiments?

    Thank you for this suggestion. The congruence of our results highlights the strengths of both approaches. As we discuss in detail for major point 4 (above), the Hi-C and OIL-PCR results both correctly identify Klebsiella as a carrier of the plasmid with CTX-M and TEM. We have now added this to the manuscript.

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  2. Evaluation Summary:

    Tracking horizontal gene transfer of mobile resistance genes is exceptionally important in the context of AMR and its burden on public health. An accessible high-throughput technique that provides an alternative to Hi-C or single-cell whole genome sequencing for associating mobile antibiotic resistance genes with their bacterial hosts in complex microbial populations is an important development for the field. The method introduced here, which relies on cellular emulsion and fusion PCR in one step, is an improvement over the previously published epicPCR. In addition, the method can be applied to other studies on complex microbial communities beyond antibiotic 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. Reviewer #2 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    The study by Diebold et al. describes a fast and scalable method that allows to link bacterial plasmids to the organisms that harbor them. The authors then go on to apply this technique to track horizontal gene transfer in an complex bacterial population originating from clinical samples. There is no doubt that the development of such methodologies for better tracking plasmidic resistance genes and following horizontal gene transfer events is very important. The authors do a good job in optimizing their method to be a one step process that has high sensitivity and relatively low error, while it can also be scaled, automated and used with multiplex primers. Subsequently, they apply this method to two clinical patient samples for which metagenomic data is available. In this case, they correctly identify expected relationships between beta-lactamase genes and specific bacterial taxa (and in particular K. pneumoniae), but also find that the same beta-lactamase genes are associated with organisms of the microbiome. With the exception of providing evidence that the association of particular genes with multiple organisms is not due to physical association of the bacteria in question, this is an interesting study putting forward a much needed technique for the study of antibiotic resistance but also other relationships in complex bacterial mixtures.

    Read the original source
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  4. Reviewer #2 (Public Review):

    Diebold et al. developed a simplified and improved version of the epicPCR method applied to environmental samples. The results section describes well how they perform their development and support the easy to use application. They clearly demonstrate that their methods could be used to screen association of specific genes to taxonomic markers in environmental microbial populations. They then apply their methods on human gut samples ranging from hospitalized patients and demonstrate demonstrate the utility of their methods to characterize the hosts of different targeted genes (notably AMR and plasmid related genes). However, most of their results are based on previous studies on the same sample. Therefore, it appears difficult to know how their method can be used on new samples. Do they need to redo a classical metagenomic analysis in order to obtain data on new samples ? What kind of metagenomic analysis is mandatory before performing their methods ? What is the depth of the metagenomic analysis ? Those are important questions as it will be clearly more expensive to perform the whole metagenomic analysis.

    The conclusion of the paper is well supported by data but the overall approach on new sample is never discussed. Moreover, the title appear somehow misleading as their methods do not allow to clearly identify plasmids but rather to link some targeted genes to taxonomic markers.

    Here are some important remarks:

    1. The supplementary Figure 6 is missing.

    2. What is the assembly used to perform their analysis (size, N50, raw reads ?)

    3. How the authors know already the structure of the Klebsiella plasmid?

    4. the authors compare the results of their sequencing with a custom database of expected sequences but what are the results if they compare it to the NCBI database ?

    5. a comparison of their results with the HiC linkage obtained in the paper by Kent et al, could clearly strengthen their claims and their results.

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  5. Reviewer #3 (Public Review):

    This manuscript is composed of two parts. The first part describes development of an emulsion-based PCR fusion method, called OIL-PCR, for matching two specific gene sequences from the same cell. In this report these are beta-lactamase genes from the V4 section of rRNA, allowing the matching of this horizontally transferred gene with its donor sequence. The second part is a demonstration project that features the use of OIL-PCR to monitor horizontal transfer of beta-lactam genes between gut bacteria from the metagenomes of two neutropenic patients. OIL-PCR was set to multiplexed class A beta-lactam genes. This is a descriptive study that largely recapitulates a previously published work on these samples showing that the relatively unstudied Romboutsia commensal genus is a carrier of these plasmid-borne genes in patient metagenomes.

    Overall, this is a well-written manuscript. Data were comprehensively analyzed with appropriate controls. The figures are excellent.

    OIL-PCR is a derived of other fusion PCR methods, especially epicPCR. There are some nice technical improvements described here, e.g efficient lysis within emulsion droplets using Ready-Lyse lysozyme. This is an incremental technical advance for a fairly niche application (where you have known target genes and are concerned about potential culture-bias) but it may be useful in particular for understanding HGT in microbiomes. There are some problems with the method that are brought to the foreground by the authors rather than quietly dropped, which is commendable. One problem appears to be that the necessary dilution for single-cell PCR reduces the taxonomic diversity of the metagenome. The only way around this to perform efficient sampling appears to be to perform multiple independent sequencing experiments and pool the results. Another feature of the system is that the accuracy falls slightly as the proportion of the target sequence in the community increases for reasons that are not discussed. However, this effect is not great (97% accuracy at 10% proportion) and most applications, the target cells will be a much lower proportion of the community.

    The results of the demonstration study on metagenomes from neutropenic patients are clearly described and provide a nicely worked example of combining this directed method with metagenome sequencing. The significance is limited but gives some descriptive hits about the mechanism of HGT between Romboutsia and Klebsiella.

    Other points:

    Unfortunately, there was no comparative test where the same samples were run against "competing" technologies (e.g sequencing of cultured beta-lactam resistant strains, epicPCR, Hi-C or single-cell) to directly compare strengths (and weaknesses) of OIL-PCR.

    As protocol development is central to this manuscript paper, and one of the main advantages claimed for OIL-PCR is ease of use, the supplement should contain a detailed protocol for control sample with a list of equipment and reagents needed and what results should be obtained. This could easily be adapted from the methods section, which is highly detailed. What is the estimated cost-per sample of this procedure and how does it compare roughly with other methods, - EPIC-PCR and culture-based?

    Line 197-198 reference needed to the Kent et al study here? What is the reason that the Hi-C results from this manuscript are not compared to the results of the OIL-PCR experiments?

    Read the original source
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