10 thoughts on “Journal Club Presentation

  1. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.

    This study investigated the role of AcrAB-TolC multidrug efflux pump in drug-resistance acquisition by plasmid transfer. They proposed this study to examine the effects of drug-resistance dissemination by horizontal gene transfer because it remains poorly understood at the cellular scale. The authors had multiple research questions regarding various aspects of drug resistance including the cellular dynamics of DNA transfer, the timing of expression of the newly acquired genes, the phenotypic conversion of the drug sensitive recipient into a resistant cell, and the effect of ambient antibiotics on these processes. To address these questions, they developed an experimental system that enabled them to visualize real-time transfer by conjugation at the single-cell level. Using this technique, they revealed the dynamics of resistance acquisition in E coli by transfer of the fertility factor–conjugation plasmid encoding the tetracycline-efflux pump TetA. They found that in the presence of antibiotics that inhibited translation, resistance acquisition depended on the AcrAB-TolC multidrug efflux pump, because it reduced tetracycline concentrations in the cell. They also found that AcrAB-TolC efflux activity can preserve resistance acquisition by plasmid transfer in the presence of antibiotics with other modes of action. Overall the study revealed the key role of a multidrug efflux complex in preserving the acquisition of drug-specific resistance by horizontal gene transfer in the presence of bacterial inhibitors

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.

    They utilized a myriad of in vitro assays such as: conjugation assays, spot assay & growth curve, ribosome-stalling assays using pDualRep2 plasmid, mass spectroscopy as well as the live-cell microscopy environments for visualization of plasmid transfer. The methodological approaches used in this study were pertinent to the proposed study. They used necessary techniques to validate their findings and support their analysis, and conclusions.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    I think the paper does a great job at explaining the ration and premise of the study. The authors proposed to examine the effects of drug-resistance dissemination owing to the global spread of antibiotic resistance which presents one of the biggest concerns in public health and research priority in microbiology. The introduction does a great job at giving adequate background and stating the problem state. They subsequently go on to express what their research questions are and to evaluate it using numerous in vitro approaches. The organization as well as the flow of the article in terms of the methodology and the progression of the results and discussion were thorough and the conclusions strongly supported. Overall, they report that AcrAB-TolC is essential for the acquisition of tetracycline resistance by horizontal gene transfer, presumably by reducing levels in the cell.

  2. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.
    Hypothesis: Based on the way the paper was written, it appears that the authors didn’t have a set hypothesis. Instead, they used their experimental system to study the timing of expression of genes obtained by conjugation, phenotypic changes in resistant cells, and the influence that antibiotics exert on the aspects of conjugation under investigation.
    Results: Using real-time microscopy, fluorescence imaging, bacterial cell culture, and in vitro conjugation assays, the authors determined that the multidrug efflux pump AcrAB-TolC plays a crucial role in acquisition of bacterial tetracycline resistance and enables this process to proceed even in the presence of antibiotics.
    Conclusion: The authors conclude that, through the present research, they were able to elucidate the role of a multidrug efflux pump in acquisition of drug-specific bacterial resistance via horizontal gene transfer.

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.
    The methods, which include live-cell microscopy, fluorescent labelling of reporter systems, and plasmid construction, are all appropriate given the author’s interest in thoroughly examining the intricacies of conjugation. Data analysis, such as the Mann-Whitney test, as well as the way in which data are displayed (i.e. violin plots) are suitable. The data indicate that the authors’ reporter system was effective and clearly illustrate the essential role that AcrAB-TolC plays in the establishment of antibiotic resistance.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.
    Introduction: In terms of overall format, the different sections of the paper flowed into one another, and I would have preferred a cleaner, more clear-cut organization. The introductory section was brief and a little generic (I would have liked more thorough coverage of the other labs’ work in the field), but it did explain the urgent health problems posed by antibiotic resistance and the clear need to learn more about this process, which established a solid rationale for the study. The authors also gave a brief explanation of the process of conjugation, though it might have been helpful to include a basic figure illustrating the concept, as well.
    Discussion: The discussion was lackluster. The authors fail to give a clear picture of what their next steps will be. Also, their statements about the implications of their findings are rather sweeping. In my opinion, they don’t explore the ramifications of their findings as much as they could have.
    Conclusions: The authors’ results support their conclusion regarding AcrAB-TolC multidrug efflux.

  3. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.

    – Despite the presence of Tc, which inhibits protein synthesis, the transconjugants were able to produce TetA efflux protein
    – Donor and WT cells that were devoid of TetR repressor experienced an enhancement of TetA-mCh production, while cells that contained preexisting TetR before plasmid entry ceased production of TetA-mCh
    – Tc-sensitive recipients produce TetA even with the Tc’s translation-inhibitory effects and thus are converted into Tc-resistant cells
    – The synthesis of plasmid-encoded proteins in the presence of Tc occurs independently of TetA activity
    – AcrAB-TolC is essential for TetA production after plasmid acquisition when Tc is present and facilitates proteins synthesis in the presence of Tc
    – Cells that have already evolved regulatory mutations could possibly acquire plasmid-mediated resistance more frequently than a WT strain

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.

    – It felt as if the investigators didn’t have one single hypothesis, but a lot of data they were trying to tie together; these results are a conga line of data they’ve been compiling for the past decade that is masquerading as a cohesively executed, intentional plan
    – Because of the sheer amount of data, it would be difficult for reviewers to properly critique and verify all claims made, and the sheer amount of information in such a limited space with a word count limit inhibiting clarification

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    – Both the introduction and conclusion were extremely short, with where the field is currently (it was at least mentioned, as well as why their research is important), how their results contribute to the field, or possible future directions
    – There is little to critique because there is very little there, which is a problem in and of itself; the introduction should justify why they are conducting the research and the conclusion should give a brief overview of what results were found and how they contribute to the field. Both this introduction and conclusion offer thin justification, likely from a restricted word count and a large amount of data that required most of the space

  4. Student Aminatta Tejan-Kamara Instructor Dr. Gewirtz Date 11/14/19
    Please answer the following. Do not exceed 1 page for your answer; you may use outline/bullet points.
    1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.

    Hypothesis: As understood from the paper, the investigators hypothesized enhanced expression of the proteins of interest could be a result of unrepressed activity of PtetA promoter as it enters the plasmid into recipient cells lacking the TetR repressor.

    Major Results: results include delta-acrAB, delta-tolC, and delta-acrABdeltatolC can “acquire the plasmid whether or not tetracycline is present.” This finding is illustrated in Figure 4A most explicitly. The methods used included the live microscopy visualization of F-Tn10 plasmid transfer, quantification of the production of TetA proteins, and multiple time course experiments to evaluate how the conjugating populations were changing in the presence/absence of tetracycline.

    Overall conclusion: Authors were able to elucidate the key role of AcrAB-TolC multidrug efflux complex “in preserving the acquisition of” resistance to tetracycline (the specific antibiotic in the study).

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.

    Authors major methods included live microscopy with real time microscopy visualization. The results demonstrate the drug efflux pump is not entirely dependent on Tetracycline’s presence, as evidenced by the results in Figure 4A, 4B, and 4C. Given the author’s noting of lack of in vivo studies regarding this research area, the real time microscopy visualization seems appropriate and serves as a robust tool. Use of controls with the cells absent of tetracycline offer a strong comparison. Thus the major results appear to be supported by the data provided. Author’s should consider correlating their in vivo data with in vitro studies to further strengthen their conclusions in both cases. Future directions should consider this approach.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    Author’s introduction is well organized and offers strong epidemiological reasons for the study given the public health concern of drug resistance. Additionally, their acknowledgement of the modest amount of investigation at the cellular level provides a strong rationale for the need to complete the studies. In general, the major results do appear to support the overall conclusion of AcrAB-TolC multidrug efflux complex’s role in multi-drug resistance.

  5. The purpose of this study is to better understand drug resistance transfer by conjugation at the single-cell level. To explore this, this study investigated the acquisition of tetracycline resistance by conjugational transfer of the paradigmatic E. coli fertility factor plasmid encoding the tetracycline-efflux pump TetA. Real-time microscopy was utilized to visualize the transfer of F-Tn10 plasmid from donor to recipient cells and then the production of TetA protein in the transconjugant cells was measured and quantified. A fluorescent parS/ParB DNA labeling system was used to visualize plasmid transfer. The parS binding site was inserted near the origin of replication site and the fluorescently labelled parB would appear in recipient cells if plasmid transfer was successful. After plasmid acquisition, TetA production was assessed via a C-terminal fusion of TetA with the mCherry fluorescent protein (TetA-mCh), which confers fully functional resistance to tetracycline. Then, F-Tn10tetA-mCh donors and Tc-sensitive recipient cells were combined together and the frequency of transconjugants was measured directly at the single-cell level as well as how long conjugation took. Both time-course and time-lapse analysis reveal that TetA-mCh levels in transconjugant cells surpass basal levels observed in donor cells. They also noted that TetA levels rise and Tc sensitivity is high prior to parallel production of TetR, which increases Tc resistance. Analysis of plasmid transfer dynamics revealed a slight delay in TetA-mCh production after plasmid acquisition in the presence of Tc indicating that protein synthesis is only partially inhibited by Tc in sensitive recipient cells. Subtraction of the contribution of Tet-A mediated-efflux showed that synthesis of plasmid-encoded proteins was independent of TetA activity in the presence of Tc. Further research on the AcrAB-TolC multidrug efflux pump proved that it is essential for TetA production after plasma acquisition in the presence of Tc because transfer of the F-Tn10tetAmCh plasmid into AcrAB-TolC mutant recipient strains resulted in no TetA being produced and Tc resistance was not developed.
    Overall, the methodology in this study seemed sound and appropriate. WT cells and controls were used throughout each experiment and it seemed that their results did align with the data presented. One thing I did not think was very clear was how they measured Tc-resistance. In the timed-resistance experiments, they stated that roughly 70% of recipient cells were converted to Tc-resistant cells within 3 hours but I wasn’t entirely sure how they measured this accurately. Also, if only 70% of the recipient cells were converted within 3 hours, what might be some explanations for the variance in conversion rates between cells? This was never expanded on or even mentioned. Furthermore, when estimating the timing of expression of plasmid genes independently of the PtetA promoter, they used a plasmid carrying the superfolder gfp gene (sgfp) but didn’t explain why exactly they used this gene in particular.
    Overall, the rationale for scientific premise is strong given that resistance to antibiotics is a problem that is still relatively poorly understood on a cellular level. The study was well written and proceeded in a logical order that was easy to follow. However, there were no separate sections, such as the conclusion, discussion, and methods sections, which I thought would’ve aided in preventing the paper from being too dense in certain parts. The authors cited numerous related and crucial previous papers, which supported their methodology and the conclusions being made. In the end, I think their results and data did support their overall conclusions. More related research needs to be done in animals specifically regarding antibiotic resistance since cellular pharmacology and physiology can differ from in vitro. A final critique is that further study was not discussed. For example, It would be interesting to know whether some of the mutations used in the study actually exist in humans or other animals at any level.

  6. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.

    – The hypothesis is that the Escherichia coli fertility factor acquires drug-resistance to translation-inhibiting antibiotics through a AcrAB-TolC multidrug efflux pump.
    – Figure 1 shows that the F-Tn10 plasmid is transferred into recipient cell from a single strand and converted to double strand DNA. Figure 2 shows the TetA production after plasmid acquisition. They show that there was full resistance to tetracycline. Figure 3 shows that the multidrug efflux pump is required for acquisition of the resistance to tetracycline. Figure 4 shows that the activity of this pump influences the drug-resistance acquisition.
    – In the conclusion the authors state the importance for AcrAB-TolC for the acquisition of tetracycline resistance in the presence of other drugs. They highlight the role of a multi-drug complex which mediates the drug-specific resistance in the presence of bacterial inhibitors.

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.

    – Methods section is in the supplementary and it would have been mice to see a summarized overview of the methods in the main manuscript. The assays used are fairly foreign to me so it was difficult to interpret their results directly from the figures, but their discussion in the text of the manuscript helped clear things up.
    – They are clear with their data analysis although they do not mention the type of statistics used in the figures, which would have been nice besides having to flip through the supplementary methods.
    – Overall, their results are substantiated by the data provided.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    – The introduction section is short, and the authors only give a brief background on the rational and previous literature on the subject before jumping right into the data. However, they do justify their current study with the previous literature cited.
    – Like the introduction, the authors seem to immediately flow into the conclusion from the results section. The conclusion section is very brief and does not give too much relationship on other findings in the field or how it is relevant translationally. However, overall their results do support their conclusion that there is a key role of a multidrug efflux complex in preserving the acquisition of resistance.

  7. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.
    The authors recognized the importance of studying the mechanism of bacterial resistance to antibiotics and identified an experimental system that enabled real-time visualization of drug resistance transfer by conjugation at the single cell level. It was hypothesized that AcrAB-TolC, a multidrug efflux pump, would play a role in drug resistance acquisition in bacteria by plasmid transfer. The acquisition of Tetracycline resistance through conjugational transfer of E. Coli fertility factor plasmid carrying insertion of Tn10 transposon was studied. The Tn10 plasmid transfer from donor to recipient cells was visualized and quantified in the production of TetA protein. It was found that AcrAB-TolC was essential for the acquisition of Tc resistance in the presence of chloramphenicol, erythromycin, and nalidixic acid. Overall the results confirmed that AcrAB-Told C activity alleviated the inhibitory effect of drugs on reactions needed for development of resistance. The authors concluded that the multidrug efflex pump did indeed contribute to the resistance acquisition of bacteria.

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.
    Overall, the methods were appropriate and the results substantiate the data reported. A variety of methods were used such as plasmid construction, fluorescence assays, and live cell microscopy. These methods were appropriate and a clear method for this study and the images were overall easy to interpret. One strength of the methods was the use of a diversity of drugs and antibiotics used. However, there is a need to separate introduction from methods and results for clarity of the paper. The data analysis were appropriate and supported the conclusion that the multidrug efflux pump played a role in drug resistance via plasmid transfer.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.
    The rationale for the scientific premise was clear: the global spread of antibiotic resistance among bacteria is one of the biggest concerns in public health, and so it is important to study its mechanism it order to improve treatment of diseases and bacterial infections. The introduction further went on to explain causes of resistance, including acquiring genes through acquisition of genes through bacterial conjugation. The discussion on potential clinical implications was lacking. It would have been beneficial to analyze what the findings from this study would imply for antibiotic treatment in humans. They did mention in the concluding remarks that antibiotic treatments that fail to prevent resistance transfer which exerting selection would increase the occurrence of resistance, but more discussion is needed. There was also no mention of potential future studies based on the results collected, such as developing drugs or ligands to act to regulate these processes. Throughout the paper, the authors mentioned established findings and literature in the field which strengthened the paper. The conclusion overall, however, seems to be substantiated by the results.

  8. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.
    Nolivos et al. revealed the dynamics of resistant acquisition of plasmid transfer by looking at the role of AcrAB-TolC, a multidrug efflux pump. The major results are that conjugation allowed for the production of the TetA efflux pump (fig. 1) by using live-cell visualization. Then they used a real-time visualization approach and observed induction of zygotes (fig.2). They also investigated the role of the efflux pump on TetA expression in wildtype and mutated cells (fig.3). Finally, they looked at how drug resistance-acquis ion influenced the regulation and activity of AcrAB-TolC (fig.4). Nolivos et al. concluded that the AcrAB-TolC pump plays a key role in preserving the ability of drug-specific resistance in the presence of inhibitors.

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.
    Method/result 1: conjugation of E.coli fertility factor (F) plasmid that carried Tn10 transponson. This Tn10 transponson is important because it encodes for the TetA efflux pump protein. The major function of this pump is to pump out Tc molecules out of the cell. Fig.1A shows work done by the plating assay which details the frequency of transconjugation of the recipients. The graph shows that in both the absence or presence of Tc the cells transconjugate. For this experiment I have some questions, for example why they decided to mix the donor and WT cells and the reason for the ratio (3:1)? I do like the explanation they provided for their “unanticipated” results. Fig.1B shows microscopy images using real-time microscopy to visualize the F-Tn10 plasmid transfer from the donor to the recipient based on the fluorescent parS/parB DNA labeling system. The transconjugant shows that the donor cells exhibit red and the recipients exhibit green and when they are transconjugated expression of both is shown. Fig 1C shows the signal of the transconjugants in the absence and presence of tetracycline at different time points. The signal seems to be overly expressed in the absence of tetracycline compared to +tetracycline. Although there is no statistical analysis to show the magnitude of the difference. Method/result 2: investigated the influence of zygotic induction using real-time microscopy.
    Method/result 3: Nolivos et al. investigated if the efflux pump had any action to protect protein synthesis and TetA expression. They tested this genetic manipulation and deleting acrA, acrB, tolC. They looked at the frequency of the transconjugants between WT and Tc cells each infected with the different genetic manipulations. In figure 3C they identify statistical significant differences between WT and all the manipulated cells.
    Method/result 4: Nolivor et al investigated effect of AcrAB-TolC regulation influenced by drug-resistance. They had the previously stated treated cells but this time a fraction of them were treated with Tc and a fraction weren’t. Overall, I don’t see a good enough range of diversity of assays (but I know nothing about the field so I might be wrong). Some results had statistics ran to make their conclusion and some did not. Also, again, im not an expert but usually that high number of cells being used is a good thing so does that same principle apply?

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    I understand that this is a Science paper but there is no real breakdown of the normal way an article is published. The authors briefly introduce the background, the idea behind the antibiotic resistance and then quickly dig into the methods/results. So, compared to articles not published in Science the introduction was very lacking of background information. They also don’t have as many references to support their ideas. The discussion did not have its own section and most of it was discussed as experiments went by. Their overall conclusion was also lacking more in depth future experimental ideas.

  9. Donald Jessup
    1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.
    Hypothesis: Drug resistant dissemination by horizontal gene transfer is not fully understood, there for the investigators developed an experimental system that enables the real-time visualization of drug resistance transfer by conjugation at the single-cell level.

    Methods/Results: 1.) Live-cell visualization of F-Tn10 plasmid transfer followed by production
    of TetA efflux pump demonstrated that within 3 hours 70.3% of the recipient cells were converted into Tc-resistant transconjugants. In the presence of Tc, which inhibits protein translation by targeting 16S ribosomal RNA, it was shown that 36.2% of recipient cells still acquired Tc resistance. Follow up experiments utilized real-time microscopy visualization of F-Tn10 plasmid transfer from donor to recipient cells and quantified the subsequent production of TetA protein in the resulting transconjugant cells as well as mCherry tagged TetA in order to monitor TetA production. These reporter systems enabled the real-time visualization of F-Tn10 conjugation and demonstrated that transcription started taking place minutes after conversion of the plasmid to dsDNA. 2.) The authors examined a mechanism for zygotic induction by using a F-Tn10tetA-mChDtetR plasmid with a deleted tetR gene and comparing TetA-mCh production levels after plasmid transfer into wild type TetR-free or into TetR-producing cells. Results of this demonstrated that zygotic induction boosted TetA gene expression and accelerates the conversion of Tc-sensitive recipients into Tc-resistant transconjugant cells capable of Tc efflux, before production of TetR can establish repression. Subsequent experiments proceeded to demonstrate that antibiotics do not directly regulate the efficiency of horizontal gene transfer, and that protein synthesis is only partially inhibited by Tc in sensitive recipient cells, and that a positive feedback cycle gradually leads to effective Tc efflux and eventually restores protein synthesis. 3.) The authors also utilized several AcrAB-TolC KO bacterial lines to demonstrate that the transporter is required for conjugation mediated acquisition of resistance in the presence of tetracycline inhibiting protein synthesis. Particularly in the experiment that showed when in the presence of tetracycline, all four mutants showed increased ability to acquire Tc resistance. Indicating that cells that had evolved regulatory mutations could acquire plasmid-mediated resistance more frequently than a wild type strain. 4.) Lastly, the authors examined the importance of AcrABTolC for the acquisition of Tc resistance in the presence of other drugs that are substrates of AcrAB-TolC. These experiments revealed that AcrAB-TolC is essential for the acquisition of Tc resistance in the presence of chloramphenicol and erythromycin (both inhibit translation), and nalidixic acid (inhibits topoisomerases). Ultimately concluding that AcrABTolC efflux activity alleviates the inhibitory effect of drugs on the reactions that are required for resistance establishment after plasmid transfer.

    Conclusion: The authors concluded that a multidrug efflux complex plays a critical role in preserving the acquisition of drug-specific resistance by horizontal gene transfer in the presence of bacterial inhibitors

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.

    This paper was clearly the work of many years of intricate experiments and detailed investigation into the transfer of resistance between bacterial strains. Overall the methodology of trying to develop a fluorescent tagging technique that would allow the authors to visualize the transfer in real time and then subsequently methodically quantifying the rate, and limitations of this resistance transfer, made for a very compelling manuscript. Overall the experiments are well controlled for and the conclusions are firmly and fairly rooted in the presented data regarding the efflux of AcrAB-TolC and the rapidly acquired resistance to tetracycline. I wouldn’t consider this a critique or a missing piece of data from the paper, but for me the paper raises the interesting question of what may this experiment perhaps look like in a bacterial culture that was more heterogeneous or representative of an approximate human microbiome. Mostly as a way of getting at the question of how might this resistance transfer look in a more clinically relevant context, messy and variable as that may ultimately be.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    The methods, rational and logic that governed this manuscript are well established in the introduction and generally straightforward to follow throughout the paper. However the actual formatting of the introduction is leaving much to be desired. I’m not sure if it’s a formatting decision by the journal or the authors but both the conclusion and the introduction just blend into the results/methods and neither is particularly exhaustive or does a passable job at rooting this paper within the field as a whole, or seems to give much room within the paper to discuss alternative interpretations, beyond a few supplemental experiments that are referenced and the occasional reference to a few prior published works the limitations of the data simply aren’t explored. I get the feeling that the impact is self-evident to the field this was published in, but it doesn’t feel very satisfying to dig through the paper and still walk away at the end only guessing at the potential relevance of the publication.

  10. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.
    H: In a cellular model, the AcrAB-TolC multidrug efflux pump plays a role in tetracycline (Tc) resistance acquisition by transfer of E.Coli fertility factor (F) plasmid in-vivo.
    R: The authors showed that a majority of recipient cells were converted to the Tc-resistant conjugates within a couple hours during conjugation of a WT F-Tn10 donor and a WT Tc-sensitive recipient strain. Then, they used real-time microscopy to visualize F-Tn10 plasmid transfer and to quantify production of TetA in the transconjugants. In order to do this the authors developed an F-Tn10 plasmid transfer reporter based on a DNA labeling system. The authors were then able to analyze the time lapse between acquisition of the plasmid and the subsequent synthesis of TetA-mCh and showed that TetA-mCh production is detected after 30 minutes. They also showed that production varied in different transconjugant cells. Using time course and time lapse analysis, the authors showed that TetA-mCh levels in transconjugant cells can pass basal levels in donor cells. They then tested the hypothesis they created a plasmid by deleting the tetR gene and compared the protein production levels after plasmid transfer into WT TetR-free or producing cells and found that protein production increased in both donor and recipient cells but was abolished in recipient cells that had preexisting TetR before plasmid entry. They then showed that plasmid transfer frequency is unaffected by the presence of the drug. By deleting genes of the efflux pump they showed that cells failed to acquire resistance in the presence of Tca.
    C: The authors concluded that tetA-mCh gene transcription and mRNA translation into proteins occurs within minutes of F plasmid being converted into dsDNA. Following the discovery that surpassing basal levels of protein were found compared to donor cells, they concluded that enhanced expression could be due to the unrepressed activity of PtetA promoter when the plasmid enters the recipient cell even in absence of the TetR repressor. Furthermore, they discussed that zygotic induction triggered by the lack of TetR repressor in the recipient, boosts tetA gene expression and accelerates conversion of recipients into transconjugant cells capable of Tc efflux. They attributed this to the fact that tetR promoter is only a portion of the strength of the tetA promoter. Later they concluded that protein synthesis is only partially inhibited by Tc in sensitive recipient cells and showed that continuous production allowed cells to produce TetA in a positive feedback mechanism. Finally they concluded that AcrAB-ToldC is essential for TetA production after plasmid acquisition when TC is present.
    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.
    M: The authors developed a model that allows the experimenter to visualize drug resistance transfer by DNA conjugation in a single cell. The use of the reporter systems was smart and allowed the authors to visualize the conjugation is real time and to conduct time-courses where snapshots of conjugation populations could be taken. I did not see the use of very many controls and many WT mice were used but no other allelic variants such as HET or KO mice were used.
    R: Statistics were appropriately provided and the data shown as well as supplemental data supported they results.
    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.
    I: The author began with a strong argument for addressing the clinical problem of bacterial resistance and how bacterial resistance can occur through DNA conjugation. They then highlighted the lack of in-vivo literature that explores how drug resistance is spread through conjugation. I unfortunately found it hard to pinpoint blatant hypothesis. Throughout the paper, the authors gave clear rationale and frameworks for how they designed their experiment.
    C/D: The authors unexpectedly found that transconjugants were able to produce TetA efflux protein despite the inhibitory effect of Tc on protein synthesis. Contrastingly, they confirmed previous literature that said antibiotics do not directly regulate the efficiency of horizontal gene transfer.

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