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  1. Reviewer #3 (Public Review):

    In the manuscript, "Dynamic persistence of UPEC intracellular bacterial communities in a human bladder-chip model of urinary tract infection" by Sharma, et al., the authors develop a bladder-on-chip model and provide evidence that this is a useful model for mimicking in vivo infections. The focus is on intracellular infection structures created by uropathogenic Escherichia coli (UPEC) seen in experimental mice infections and "real" human infections; such structures have been most extensively characterized in mouse models for obvious reasons. The authors focus on three key aspects: development of a structure known as an intracellular bacterial community (IBC), the neutrophil response to infecting UPEC, and the bacterial response to antibiotic treatment. There is a minor point about the ability to apply mechanical stretch to the model to mimic bladder filling and voiding.

    In my assessment, key strengths of this work are:

    1. Integration of both epithelial and vascular endothelial cell types, allowing for multiple fluid spaces and studies of neutrophil migration

    2. Ability to apply mechanical stretch to the entire system to mimic changes in bladder volume

    3. Extensive microscopic characterization of the model (a key feature enabled by this system) including live microscopy, immunostaining, and electron microscopy

    I believe there is one key underlying issue with this paper: as a report on the technical development of a new system / device / technique, the authors have what amounts to a very strong hypothesis, namely that their new system is a good model for the in vivo infection. This leads to a general bias in both the presentation and, in my opinion, the interpretation of the data, to make the system sound "as good as possible". Key manifestations of this bias and overinterpretation include:

    1. The immediate interpretation of all intracellular structures as IBCs.

    2. The immediate interpretation of all data in Figure 2 as neutrophil swarms and NETs.

    3. Some odd behaviors in response to ampicillin, which should not penetrate host cells and has been shown using the same cell types to not affect intracellular UPEC.

    4. The claim that a 10% linear change in dimension is "physiologically relevant" and "a significant proportion" of that seen in vivo.

    To clarify point 1 (which applies as well to point 2), IBC is an abbreviation for "intracellular bacterial community", and these were first described in mice. There has been very sparing molecular characterization of IBCs, which makes a morphological classification very tricky - I believe the field generally thinks that IBCs refer to a specific structure that is formed (at least) in mice and humans in vivo. Somewhat similar structures have been seen previously in vitro but rightfully are more carefully described with different terms or as "structures resembling IBCs". I think similar care needs to be taken with this model as well.

    Overall, the authors have done quite a complete job in characterizing their model and have good data to argue for a morphological similarity to key steps that have been previously described to happen in vivo. I believe they get ahead of themselves both in data interpretation and in the writing of the manuscript, which leads to some oddness where it seems the authors begin to talk as if their model has already been validated. This occurs throughout the manuscript in the use of the IBC abbreviation and also largely in the section on neutrophil responses (in particular swarms and NETs). There are occasional sentences where the appropriate care is taken (i.e. that the data is being collected to argue that the structures seen are indeed NETs), but this is interspersed with writing that is assuming the point is already proven (for example, see lines 286 (appropriate) and 287-289 (not); and 471-476 (appropriate), 477-479 (not), 481-483 (appropriate)).

    Regarding the ampicillin data, the odd behaviors are:

    1. Apparent elimination of intracellular UPEC (particularly for large collections)

    2. Apparent indifference for some intracellular UPEC (they continue to grow)

    3. Ampicillin is generally thought to not cross host membranes, and in Blango & Mulvey 2010 it does not affect UPEC harbored within 5637 cells. The authors collect #1 and #2 under "dynamic heterogeneity" and then claim in the discussion that they can "realistically model antibiotic treatment regimens". Given these discrepancies listed above, I do not believe they can yet support this claim.

    Finally, the ability to apply mechanical stretch is only used in one pilot experiment at the end, producing a suggestive result (that UPEC burden increases when a duty cycle of stretching and relaxing is used). This is a key advantange of their model that gets a proportionately larger share of attention in the introduction and discussion. It also may provide an explanation for the ability of ampicillin to enter the host cells, or to access intracellular bacteria (through vesicular uptake during contraction, as UPEC themselves are thought to do).

  2. Reviewer #2 (Public Review):

    Sharma et al established a bladder on a chip model for studies of E. coli infection using a co-culture HTB9 bladder epithelial cells and primary human bladder microvascular endothelial cells in an organ-on-a-chip device. The two cell types expressed cell-specific markers when cultivated on-a-chip. Linear strain was applied to the sides of the device up to 19% to mimic stretching during bladder filling. The bladder chip was perfused with the diluted human urine during the experiments. The authors also observed formation of neutrophil extracellular traps by neutrophils in the infected bladder chip. They also demonstrate that the planktonic bacteria are eliminated upon application of antibiotics on a chip, with intracellular bacteria retaining the ability to grow after a lag period. The strength of the system is its fine imaging capability. It is necessary to consider if another antibiotic would enable clearance of intracellular bacteria.

  3. Reviewer #1 (Public Review):

    The complexity of the infection model developed by the authors is to be praised as it allows the dissection of host-pathogen interactions with multiple players coming together, namely human epithelial cells, endothelial cells and neutrophils, UPEC, urine, antibiotics and mechanical forces at play during bladder filling and micturition. This is truly a tour de force and should provide the authors (and other labs potentially able to recapitulate it) with an unprecedented model to study UTIs and their response to antibiotics. Notably, authors have been able to document the formation of NETs in response to UPEC infection in this model. One small caveat was the choice of antibiotics used to treat the infection in their model. Is Ampicillin really a drug of choice, both because of its inability to reach intracellular niches and it not being a drug of choice in the clinic?

  4. Evaluation Summary:

    Reviewers value the development and characterization of a bladder-on-chip infection model for recapitulating the multiples factors involved in UPEC driven UTIs. Notably, it consists of human bladder epithelial cells, bladder microvascular endothelial cells, neutrophils and urine that are also subjected to mechanical changes mimicking those occurring during bladder filling and micturition. This model is a lot more complex than in vitro tissue culture models and more amenable to analysis such as imaging than animal models and therefore constitute a distinct advance for in vitro modeling of UTI that has potential to reveal key aspects of UTIs and reasons for the difficulty to clear these infections with antibiotics.

    (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.)