Reproducibility Project: Cancer Biology we published a Registered Report  that described how we intended to replicate selected experiments from the paper “Intestinal Inflammation Targets Cancer-Inducing Activity of the Microbiota” . Here we report the results. We observed no impact on bacterial growth or colonization capacity when the polyketide synthase (pks) genotoxic island was deleted from E. coli NC101, similar to the original study . However, for the experiment that compared inflammation, invasion, and neoplasia in azoxymethane (AOM)-treated interleukin-10-deficient mice mono-associated with NC101 or NC101pks the experimental timing of the replication attempt was longer than that of the original study. This difference was because in the original study the methodology was not clearly stated and likely led to the increased mortality and severity of inflammation observed in this replication attempt. Additionally, early death occurred during AOM treatment with higher mortality observed in NC101pks mono-associated mice compared to NC101, which was in the same direction, but more severe than the original study . A meta-analysis suggests that mice mono-associated with NC101pks have higher mortality compared to NC101. While these data were unable to address whether, under the conditions of the original study, NC101 and NC101pks differ in inflammation, invasion, and neoplasia this replication attempt demonstrates that clear description of experimental methods is essential to ensure accurate reproduction of experimental studies.As part of the  Reproducibility Project: Cancer Biology (RP:CB) is a collaboration between the Center for Open Science and Science Exchange that seeks to address concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology (The  biology . For eac biology  for a 20-/-Il10 ), infectious , or Salmonella typhimurium), or a combination of these  mice and E. coli mono-association to produce a background of chronic inflammation followed by six weekly injections of AOM to induce neoplastic transformation. Using this inflammation-induced CRC model, Arthur and colleagues reported that germ-free mice mono-associated with the commensal mouse adherent-invasive E. coli strain NC101 developed invasive mucinous carcinomas which did not occur in mice mono-associated with Enterococcus faecalis, another colitis-inducing bacterial strain (pks) pathogenicity island that encodes the biosynthetic machinery for synthesizing the genotoxin colibactin , without altering colonic inflammation . The model used in that study was one of a group of related models that are commonly used to study the role of inflammation in colon carcinogenesis . These ml strain . NC101 hlibactin . Mono-asammation .pks-harboring E. coli strains to enhance tumorigenesis. Similar observations were observed using Min/+APC mice , and summarized the current evidence for these findings . Since tn/+ mice , germ-fr-/- mice , or a AO-/- mice . A follo-/- mice .The outcome measures reported in this Replication Study will be aggregated with those from the other Replication Studies to create a dataset that will be examined to provide evidence about reproducibility of cancer biology research, and to identify factors that influence reproducibility more generally.E. coli NC101 strain and an isogenic pks-deficient (NC101pks) strain as the original study  mice in their hepatic metabolism. Because AOM is a hepatotoxin in addition to a carcinogen, its toxicity may be altered in mice without a normal microbiota  were mono-associated with either the NC101 or NC101pks isolate described above, treated with AOM, and then assessed for colonic inflammation and tumorigenesis. The original study also reported cohorts of mice that did not receive AOM treatment (assessed at 12 weeks), or received AOM treatment and were assessed at 14 weeks. These additional cohorts were not included in the design of this replication attempt. Furthermore, the number of mice required for this study to have sufficient power to detect the originally reported effect sizes were determined a priori and took into account the number of anticipated animal deaths that would occur prior to the 18 week assessment based on the originally reported survival rates. To summarize, bacterial colonization was confirmed 4 weeks after mono-association, after which AOM was administered weekly for a total of 6 injections, and then mice were monitored for 18 weeks after the last AOM injection , we did not achieve the planned number of animals at the end point of 18 weeks post-AOM treatment. Only 11 mice survived to 17-18 weeks after AOM treatment , while a total of 25 mice survived 14 weeks post-AOM treatment or later. During the course of the entire study, there was a median survival of 154 days (range: 31-176 days) and 57 days (range: 29-188 days) for NC101 and NC101pks mono-associated mice, respectively, which was not statistically significant (log-rank (Mantel-Cox) test; p = 0.0608). Regardless of mono-associated bacterial strain, mortality was highest during AOM treatment; however during this interval, mortality was greater in mice mono-associated with NC101pks compared to mice mono-associated with NC101. In the NC101pks group, fewer than half the animals survived beyond the last AOM treatment. Importantly, there were similar levels of colonization capacity in vivo for both strains of bacteria , male mice became overrepresented during the course of this study: 14 weeks post-AOM treatment ; 17-18 weeks post-AOM treatment . In the original study 4 female and 8 male mice were mono-associated with NC101 and 8 male mice were mono-associated with NC101pks  of which 14 mice  survived to 18 weeks after mono-association . This corresponds to a median survival of 57 days for NC101pks while the median survival for NC101 mono-associated mice could not be determined since more than half of the animals were still alive at 18 weeks following mono-association. An exploratory analysis to compare the survival distributions between the two groups during this timeframe was statistically significant (log-rank (Mantel-Cox) test; p = 0.0230). The original study reported that the absence of the pks island had a small, but not statistically significant effect on mouse survival (NC101: 75% (9 of 12 mice); NC101pks: 62.5% (5 of 8 mice)) ) . When copks = 3) had massive acute hepatic necrosis that appeared severe enough to have caused death  and was present in all mice that were examined histologically 9 weeks or more after mono-association. Most mice also had typhlitis, which was less severe than the colitis. Inclusion of AOM-only controls could have indicated any increased susceptibility of the mice used in this study to AOM and should be considered in the experimental design of future studies.pks at 14 weeks; NC101pks at 14 weeks), one with typhlitis and dysplasia (NC101pks at 19 weeks), and three with colon adenocarcinoma . Furthermore, five mice (between 19 and 27 weeks after mono-association) had anal squamous cell carcinoma in addition to colon adenocarcinoma  (Colon adenocarcinoma was first detected in mice that died 8 weeks after AOM treatment (17 weeks after mono-association) and was present in all mice euthanized 13 weeks or more after AOM treatment. Notably, a few mice died at time points close to the time that mice were harvested in the original study: one with typhlitis (NC101 at 14 weeks), two with severe colitis (NC101pks = 2) .pks. In the few cases (10 of 24 mice) where histologically detectable non-neoplastic tissue was present between neoplastic foci, an attempt was made to enumerate individual tumors in histologic sections (pks mono-associated mice (median count of 2). Because individual tumors could not be distinguished, tumor counts cannot be directly compared to the original study where the absence of the pks island resulted in a statistically significant decrease in macroscopic tumor burden , without an impact on tumor size  and invasion scores , but not inflammation scores  18 weeks after mono-association (t test), which is not appropriate since the mean cannot be defined .Colonic inflammation and tumorigenesis were scored as far as possible using the same scoring criteria as the original study  while taociation . Althoug defined , similarpks would have been masked by the greatly increased severity of the lesions. Additionally, over time it is possible the products of excessive inflammatory responses (e.g. reactive oxygen species), which promote tumorigenesis, become more important than pks status. Thus, it is possible that the experimental timing in this mouse model is crucial for differentiating the outcomes of NC101 and NC101pks. While subjective differences in histologic interpretation could also account for differences between the studies  and sample size.We performed a meta-analysis using a random-effects model, where possible, to combine each of the effects described above as pre-specified in the confirmatory analysis plan . We exclpks strains was not conducted since the original study reported a single growth curve for both strains. Comparing the original and replication results, the original value reported in pks was within the 95% CI  and implies the null hypothesis that the survival distributions for the two cohorts are the same, can be rejected.The comparison of the overall survival distributions between NC101 mono-associated mice compared to those that were mono-associated with NC101al study . Importaanalysis  of theseThis direct replication provides an opportunity to understand the present evidence of these effects. Any known differences, including reagents and protocol differences, were identified prior to conducting the experimental work and described in the Registered Report . HoweverAs described in the Registered Report , we atteE. coli. NC101, isolated from the feces of an inbred 129S6/SvEv background mouse raised in SPF conditions , and NC101pks (8 CFU prior to inoculation into Luria-Bertani (LB) broth. Bacteria were grown at 37˚C in room air with shaking for all experiments.101Δpks , were shE. coli strains that were grown overnight (12–16 hr) were used to inoculate 10 ml of LB broth at 1:5, 1:50, 1:500, and 1:5000 dilutions. Starting at the time of inoculation, cultures were plated in a 96 well plate in technical triplicate and were measured every 30 min at 600 nm absorbance using a microplate spectrophotometer. Plates were maintained at 37˚C during the assay. LB broth was used to determine the background, which was subtracted from the readings. Technical repeats were averaged for each biological repeat. To summarize the growth characteristics (e.g. doubling time), values for each biological repeat were fit to the standard form of the logistic equation common in ecology and evolution using the Growthcurver R package (SCR_001905), version 3.4.4 ( package  and R soon 3.4.4 .SCR_014241). PCR reactions were performed using primers specific for the 5’ and 3’ end of the pks island, colibactin, and 16S rRNA, with sequences listed in the Registered Report , 0.05 mM dNTPs, 0.05 µM forward and reverse primers, and 2 µl DNA diluted in water. A negative control without DNA was also included. PCR cycling conditions were: 1 cycle 95°C for 5 min – 35 cycles (or 27 cycles) 95°C for 45 s, 56°C 45 s, 72°C 45 s – 1 cycle 72°C for 10 min. PCR reactions were run on a 1.5% agarose gel to visualize if a product of expected size was produced.Bacterial DNA was isolated from overnight cultures using a DNeasy Blood and Tissue kit according to manufacturer’s instructions . Fecal material was processed for DNA extraction by resuspending pellets in lysis buffer supplemented with 20 mg/ml lysozyme, incubated at 37˚C for 30 min and then supplemented with 10% SDS and 350 µg/ml proteinase K. DNA was extracted with a DNeasy Blood and Tissue kit according to the manufacturer’s instructions. DNA was quantified using a NanoDrop spectrophotometer . PCR was performed on a MJ Mini Gradient Thermal Cycler  and Opticon Monitor software (RRID:d Report . Reactiohttps://osf.io/fnu62/) using the NEBNext Ultra DNA Library Prep Kit for Illumina  and sample-specific barcoding. Library preparation and sequencing were performed at the Center for Microbial Systems at the University of Michigan. Samples were evaluated for contamination and excessive low quality sequence with FastQC (RRID:SCR_014583), version 0.11.5 (SCR_011848), version 0.36 (De novo genome assemblies were generated for each sample by running SPAdes (RRID:SCR_000131), version 3.5.0 (pks was ordered relative to NC101 using ABACAS (RRID:SCR_015852), version 1.3.1 (SCR_014732), version 1.11  according to the manufacturer’s instructions and prepared for sequencing on an Illumina MiSeq instrument   using the short-read Burrows-Wheeler Aligner (BWA) (RRID:SCR_015853), version 0.7.13 (SCR_006525), version 1.130 (http://broadinstitute.github.io/picard), and (3) calling variants with SAMtools and BCFtools (RRID:SCR_005227), version 1.2 (SCR_001876) VariantFiltration, version 3.3.0  (https://osf.io/jgqdb/) was used to filter out single nucleotide variants that were: (1) < 5 bp in proximity to indels or (2) < 10 bp in proximity to another variant.Variants were identified by: (1) mapping filtered reads from NC101n 0.7.13 , (2) dission 1.2 . Variant< 0.025) . In addipks, bi-directional BLAST queries were performed between the contigs in the genome assemblies. Regions found to be unique to either genome were verified by mapping reads using BWA and visually verifying that no reads map to putative unique genomic regions using the Artemis Comparison Tool (RRID:SCR_004507), version 13.0.0  to the Germ-Free & Gnotobiotic Mouse Facilities at Michigan University. Mice for this study were born and raised in GF isolators until they reached the age of 7-12 weeks. GF status was verified by bacterial culture, Gram stain, mold trap, and 16S bacterial PCR. Gram stain and bacterial culture were performed at every isolator entry. Mice were aseptically removed from the isolators, randomly assigned to be mono-associated with NC101 or NC101pks, and housed in sterile isocages (Tecniplast), where they stayed throughout the study. After the mice were moved to the isocages, gnotobiotic status was verified weekly by Gram stain and bacterial culture, while mold traps were monitored daily to confirm GF status. Early in the study a few isocages were contaminated and had to be removed from the study. This did not affect the other isocages which remained gnotobiotic.Germfree (GF) pks: female=23, male=22). Mice of the same sex and mono-associated with the same bacteria strain were caged together (2-4 mice per isocage). Mice were mono-associated by oral gavage and rectal swabbing with 200 µl of an overnight log phase bacterial culture at a concentration of 2x109 colony forming units (CFU)/ml. Gnotobiotic status was verified weekly by Gram stain and culture of fecal contents. For culture, sterile swabs were used to transfer fecal material to sheep blood agar plates, which were incubated at 37°C under aerobic or anaerobic conditions. For Gram staining, swabs with fecal material were transferred to glass slides and spread evenly in a thin layer. Slides were air-dried, heat-fixed, and Gram stained using a BBL Gram Stain Kit according to manufacturer’s instructions . Throughout the experiment, mice were observed at least once daily and weighed weekly. Moribund and dead animals were necropsied unless autolysis precluded any interpretation. Mice were offered Purina Lab Diet 3500  and sterile water ad libitum. They were housed on Tek-Fresh bedding (Envigo) and offered Enviro-dri nesting material  as enrichment.A similar distribution of male and female mice were assigned to both strains  were initiated and continued until mice received six injections. The same lot of AOM was used for the entire study with 25 mg/ml aliquots stored at −80˚C until use. One vial was used for each injection day and any remainder discarded to avoid unnecessary freeze-thaw cycles. Mice were euthanized and necropsied 18 weeks after the sixth AOM injection as prespecified in the Registered Report , or when600 readings. Cultures were adjusted to the desired density on the OD reading and mice were mono-associated. For precise determination of CFU/ml, an aliquot of the culture was quantified by serial dilution (10 fold dilutions in LB broth). 100 µl of each dilution were plated on LB agar and incubated at 37°C for 24 hr under aerobic conditions. On plates with discrete colonies, the number of colonies were counted and results were expressed as CFU/ml of contents.To quantify cultures for mono-association, bacteria were cultured overnight (12–16 hr) at 37°C in LB broth to log phase growth and CFU/ml was estimated based on ODTo quantify colonization of mice, samples of feces were aseptically collected into pre-weighed, sterile tubes (average weight of feces was 0.05 g). Samples were resuspended as a slurry in 1 ml sterile LB broth and serially diluted (10 fold dilutions in LB broth). 100 µl of each dilution were plated on LB agar and incubated at 37°C for 24 hr under aerobic conditions. On plates with discrete colonies, the number of colonies were counted and results were expressed as CFU/ml of contents.https://osf.io/pm5xa/.Mice were sacrificed at the indicated time points and colon , distal) and cecum, liver, spleen, and any gross lesions were collected for histological evaluation. Colons were blindly examined macroscopically for tumors by a board-certified veterinary pathologist. Tissues were fixed in 10% neutral buffered formalin for 24–48 hr, with colon tissue Swiss-rolled from the proximal to the distal end. The fixed tissue was embedded in paraffin, sectioned at five microns, and stained with hematoxylin and eosin (H&E) as described in the Registered Report . IndividScoring of inflammation was based on a study by in situ, also referred to as small non-invasive adenomas or individually transformed crypts , version 3.4.4 (https://osf.io/y4tvd/). Confirmatory statistical analysis was pre-registered (https://osf.io/yt9ki/) before the experimental work began as outlined in the Registered Report th percentile of the normal distribution (https://osf.io/2raud/). The original study data presented in a priori from the published Figure by estimating the value of each symbol based on the scoring criteria described in the original study methods and shared by the original authors. The data were published in the Registered Report   that was used to compose this manuscript, which is a reproducible document linking the results in the article directly to the data and code that produced them  Authors established colonization of germ free 2) AOM injection in the colonized mice induced colorectal tumorigenesis as in original paper.E. coli genome did not affect colonization and bacterial growth of E. coli in germ free mice.3) Similarly to Arthur et al., deletion of PKS island in 4) Authors conclude that ΔPKS induces similar level of inflammation  and similar level of tumorigenicity  in comparison with PKS+ colonized animals.5) Authors claim that overall inflammation and other histopathological observations were more severe in the replication attempt.6) Overall mortality of mice throughout the experiment was much higher in the replication attempt.7) Meta-analyses suggest that there is no difference between ΔPKS and PKS+ colonized mice, contrary to the original paper by Arthur et al., 2012.8) Authors hint at the conclusion that the main observation of Arthur et al. paper is not reproduced  but also conclude that results seen in replication attempt may be confounded by the increased severity of inflammation and mortality of animals during the experiment.Overall authors performed all of the experiments proposed. Data analysis is solid. This reviewer is not an expert biostatistician, so no professional comments on the statistics part can be made.Specific comments:1) Mortality seen on Figure 2A. These curves may represent two different types of mortalities (causes)- AOM induced mortality early on and then inflammation and/or tumor load induced mortality later.Early mortality in the Replication experiment is clearly higher than in Arthur et al. (>50% vs less than 30%).What is presented on Figure 2B may be very similar to the data on Supplementary Figure 10 by Arthur et al., , if mortality between day 0 and 10 is still attributed to the action of AOM. Then long time there is no mortality (days 20 to 80) in both Replication experiment and in Arthur et al. Then mice in Arthur et al., seemingly start to die, but they are immediately collected , and experiment stopped, so we do not know what happens to mortality. In Replication experiment past day 90 mortality starts but mice are euthanized according to symptoms or dying, so there is an impression of high mortality.2) One important discrepancy here is that in Arthur et al., 14 wk and 18 wk point mice were collected when they still were not dying, indicating that probably there is still a room for a difference in inflammation and tumor load/invasion.In Replication attempt, it seems from the Figure legend that mice were mostly collected and "declared dead" when they were already sick (right thing to do in terms of following IACUC regulation) but one could think that by the time PKS+ and ΔPKS mice are already equally very sick, tumor load and inflammation may be already too high and indistinguishable. Indeed, authors note that overall role of inflammation, tumor load and mortality in their experiment was higher than in initial experiment.3) Another thing noted during initial planning of experiments and review of Registered Report is that AOM quality may vary. Indeed, different lots of AOM may have different amount of "acting" AOM and sometimes it”s too much- more toxicity/mortality and more tumors in residual mice or poor decayed AOM- more toxicity/mortality and less tumors at the end. Seemingly, in Replication report higher mortality (presumably AOM induced) and higher tumor load is seen.4) Since AOM metabolism and potentially bacterial and liver metabolism may be involved, the diets used for germ free mice should be compared and that should be discussed5) Using Chi- values for tumor numbers in Replication study when tumors cannot be enumerated (why?) further complicates the interpretation.6) Another important caveat which should be acknowledged also stems from high mortality. When mortality is that high, there may be a natural selection for mice with lower inflammatory response and lower tumor load- and the mice where potentially there was (could be) a difference have been kept for too long under these conditions of inflammation and AOM lot. This is not very scientific argument, I know, but after this Replication attempt is published, the field will need all of the possible scenarios/explanations discussed.7) Inflammation is known to promote cancer. PKS+ bacteria is proposed to be more carcinogenic than PKSΔ. However, under the conditions of stronger inflammation (observed in Replication attempt), Inflammation arguably can become more important than PKS status. PKS is genotoxic, but so are the products of excessive inflammatory responses . One could argue that under stronger inflammation the presence or absence of PKS becomes less relevant as for its effect towards tumor progression. Therefore, comparison of this (Replication) and Arthur et al. studies with regard to tumor characteristics only makes sense if the same levels of inflammation are achieved.In my view, for these experiments the technical attempt to reproduce the results has been attempted but based on the confounded data it is difficult to judge whether important conceptual results are reproduced or not (i.e. whether PKS+ is superior at tumor progression).E. coli induces more tumors and more aggressive tumors; than ΔPKS controls.The only thing which can be concluded, in my view, is that during the Replication attempt, the team did not achieve the same levels of AOM toxicity/action, the same levels of inflammation and tumor load (more); and observed higher mortality. Under these circumstances it is still not clear whether PKS+ Reviewer #2:The authors of the this replication study of the paper by Artur et al., discuss in detail all experiments performed in their study based on the initial registered replication plan and also justify all changes done compared to that protocol. They have also included in their Introduction all relevant articles published since the Registered Report was published.The major experimental findings of the study are the following:E. coli strains NC101 and NC101𝛥pks  and observe that pks deletion does not affect the growth curve, in agreement to what reported by Arthur et al. Therefore, the deletion of the pks island does not affect E. coli growth in vitro.1) In Figure 1 the authors compare in vitro the growth of E. coli NC101𝛥pks bears a genetic deletion of the pks island and shows no other genetic variants or indels compared to the NC101 strain. .2) The authors verify that E. coli strains NC101 and NC101𝛥pks display a similar growth in vivo in monocolonized germ-free -/-Il10 mice, as assessed by CFU/ml in the feces. .3) The authors validate that -/-Il10 mice that are mono-associated with E. coli NC101 or E. coli NC101𝛥pks. Extensive lethality was observed in mice monocolonized with both strains, starting after the first AOM injection. No statistically significant differences were observed between mice colonize with the two strains in terms of survival over the course of the experiment.4) In Figure 2 the authors compare the survival curves of germ-free 5) In Figure 3 the authors count the number of tumors/mouse in surviving mice in which individual tumors could be distinguished and also evaluate the extent of inflammation in them.6) In Figure 4 the authors present the results of metanalyses of their results and the ones by Arthur et al., as described in the registered replication plan.Major points:E. coli strains NC101 and NC101𝛥pks in inducing intestinal inflammation and tumor formation in monocolonized GF -/-Il10 mice in order to evaluate the reproducibility of the experiments published by Arthur et al. For this comparison to be performed the kinetics of the model should be similar to what was reported by Arthur et al. In the present study mice mono-colonized with both E. coli strains display a much higher lethality and an exacerbated phenotype by histopathology as compared to the report by Arthur et al. A very low number of mice met the endpoint of the study. Because of these issues no conclusions can be drawn regarding the potential of E. coli NC101 vs NC101𝛥pks to induce intestinal inflammation and tumor formation in monocolonized GF -/-Il10 mice.The major aim of the present study is to compare the potential of the In the Abstract the authors first mention that "Mono-association […] resulted in similar levels of intestinal inflammation and tumorigenesis; whereas the original study reported decreased tumor multiplicity […]" and secondly, they mention that this replication study showed more severe histopathological observations and a much lower survival rate compared to the original study. This structure of the Abstract is misleading to the reader. The strong differences in the kinetics of the experiment compared to the original report and the lack of power of the current study to assess the reproducibility of the original findings should be mentioned first. The observations regarding tumor number and histopathological features could be discussed as indicative of a lack of difference in the context of a severely exacerbated disease course.E. coli NC101 -/-Il10 mono-colonization model is characterized by important technical limitations that should be carefully controlled in future studies. A careful standardization of the protocol/disease course is required in individual laboratories before comparing different E. coli strains. This should lead to low mortality rates and distinguishable individual tumors at the endpoint. Including non-colonized AOM-injected-only control mice and mono-colonization-only, AOM-untreated control mice is essential for the correct interpretation of results in this model. Such factors should be considered for the experimental design of future studies.In my opinion, the major conclusion of this study is that the AOM/Additional points:The comparison of the death rates between this study and Arthur et al. is vague in this part of the manuscript despite the fact that this is the major limitation of this replication attempt. The authors should clearly mention the percentages of mice meeting the endpoint in the two studies.Mortality was highest during AOM treatment independently of the bacterial strain used whereas mice started dying right after the first AOM injection. The interpretation of these observations is difficult because of the absence of AOM-only controls: lethality over the injection period may be an effect of AOM toxicity alone or of AOM in combination with the effect of E.coli. It may also be an effect of the injection process itself . This is an additional technical variable that should be mentioned in the Discussion.The authors mention that mortality during AOM injections was greater in mice colonized with NC101𝛥pks. Is this difference statistically significant? This should be mentioned and if significant further discussed.The authors discuss sex-related differences which may confound the differences observed by Arthur et al. where 4 female and 8 male mice mono-associated with NC101 and 8 male mice mono-associated with NC101𝛥pks were analyzed. What is the result of these analyses if only male mice are considered? (n = 8 vs 8)?The study is not powered enough to draw conclusions from the histopathological analyses because of the small number of mice resulting from an exacerbated disease course in the experiment.Figure 2A: Log-rank test not shown in the legend.Reviewer #3:In this replication study, the authors had conducted the experiments under the conditions as close as possible to the original study; correctly reported everything they observed in the study including both the experiments that they were able and unable to replicate; and reasoned that they were unable to reproduce the intestinal inflammation and tumorigenesis results may be due to the overall disease development in this replication study was much more severe than the original study.-/-Il10mice were derived from the same germ-free colony used in the original study) , and the used AOM dose, 10 mg/kg, was the same as the original study . AOM administration  had resulted in a significantly higher mortality rate in this replication study compared with the original study. As cited by the authors , a previous study  showed that 33 mice were treated with 10 mg/kg of AOM, and "no premature loss of mice" was observed at "six months after the first AOM dose". This appears in line with the original study and indicates that 10 mg/kg of AOM should not cause substantial mortality rate; in contrast, AOM administration has resulted in a significantly higher mortality rate in this replication study, which could be due to the differences in AOM lots, mouse facilities, microbiota, etc. It appears reasonable that the difference in the effects of NC101 to NC101 Δpks on the intestinal inflammation and tumorigenesis, as reported in the original study, could not be replicated, with the more severe disease development in this replication study. However, in order to firmly evaluate the reproducibility of the original study, especially the effects of NC101 to NC101 Δpks on the intestinal inflammation and tumorigenesis, the experimental conditions (such as the administrated AOM dosage) could be adjusted in this replication study to get enough animals surviving through 18 weeks for the further analyses.It appears that the authors of this replication study had tried the best to conduct the experiments under the conditions as close as possible to the original study. For instance, bacterial strains were shared by the Arthur Lab , germ-free Reviewer #4:in vivo, no difference in detecting the pks island by PCR and sequencing. The authors also include a meta-analysis of the current and former studies. However, the main finding that E. coli pks promotes cancer could not be reproduced. Significant differences in experimental timeframe likely underlie this observation – see below. I also have a significant concern about mention of "bacterial contamination" and the extent of AOM toxicity.This current study is a replication of Arthur et al., 2012. The current study was able to reproduce many findings of the original 2012 study: no differences in bacterial growth and colonization between strains, no difference in histologic inflammation 1) In both studies, colonization was established for 4 weeks, after which AOM was administered for 6 weeks. However, the current study evaluated survival and tumorigenesis 18 weeks after the last (6th) AOM injection, whereas the original 2012 study evaluated survival and tumorigenesis 18 weeks after colonization. This equates to 10 weeks longer in the current study, which is 50% greater time than in the original, and could explain why no difference in tumorigenesis was observed. The authors in fact indicate that it was difficult to detect differences in tumorigenesis because it had advanced so far and many mice did not survive to this time point. This substantial difference in experimental timing – 18 weeks (2012 study) and 28 weeks (current study) – is a major flaw in this replication study. This difference should be clearly stated in the Abstract and manuscript text. Further, I”m not sure if the meta-analysis in Figure 4 is appropriate with such differences in time frame between the two studies.-/-Il10 studies. Perhaps the current authors injected mice with a very concentrated AOM solution? Survival curves from the original 2012 paper do not indicate this extent of toxicity. I recommend the authors amend their Abstract and manuscript text to indicate how different these results were between the current and former studies.2) Potential AOM toxicity: It is concerning that AOM treatment killed 30-70% of the mice, simply during the 6 week injection period. I am not aware of this extent of toxicity in AOM/DSS or AOM/3) Figure 2 figure legend mentions "animals where bacterial contamination was detected (7 out of 84) were censored". What does this mean? Does this mean that some animals housed in gnotobiotic (mono-associated) isolators became contaminated? This is highly concerning. It also raises the possibility that these mice were also infected with other non-bacterial microorganisms, such as virus or fungus. Any contamination could alter results. Potential contamination is only mentioned in this figure legend and does not appear to be mentioned in Results section or Materials and methods section. The authors must explain in greater detail what this "bacterial contamination" means – both to this reviewer and to the readers.eLife. Your revised article has been favorably evaluated by Wendy Garrett (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Replication Study: Intestinal inflammation targets cancer-inducing activity of the microbiota" for further consideration at The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:The authors provided a highly responsive revision of the original paper. In re-reviewing Eaton, 2015 (study plan) and the original Arthur et al., paper (2012), we agree that the time line for the mouse experiments proposed in Eaton, 2015 was clear whereas the time line between bacterial inoculation, AOM administration and mouse harvest was hard to discern in the original paper. Besides the importance of clearer time line delineation in original manuscripts, this highlights flaws in the review of the original study plan, certainly a distributed responsibility in which several reviews failed to raise any questions.Thus, in the review of the current manuscript, there a number of places where the text should be modified to be clearer and more detailed as listed below.E. coli NC101 was detected in the lumen of Il10 KO mice relative to WT controls. This is Figure 1I in Arthur et al. In this experiment, conducted at 20 weeks after transfer to SPF conditions, only the presence of E. coli was examined, not NC101 which would have required specific testing for pks or colibactin. NC101 is referred to but, not specifically tested for at this juncture in the Arthur et al., paper.1) Introduction. The authors state incorrectly that in Arthur et al., a 100-fold increase in -/-Il10 mice mono-associated with E. coli NC101 or NC101𝛥pks”. It was difficult to find wording in the Arthur et al. paper that clearly delineated whether harvest occurred 18 weeks from monoassociation vs 18 weeks post-AOM (wording was “14 and 18 weeks with AOM”) and better definition was missed by several reviews of the Eaton et al. plan including, even perhaps the original authors, as such the following wording change is suggested: “based on methods derived from the original study and not corrected on review of Eaton et al.” as this may represent a more accurate presentation of what occurred.2) Subsection “Intestinal tumorigenesis and inflammation of germ-free Δpks 45 mice) since the reader can‘t interpret the mouse survival numbers without this information.3) Need to insert the starting N of mice  Please expand “suggesting bacterial load was not a factor in the survival differences” as follows: “suggesting while bacterial load was not a factor in the survival differences, host:bacterial interactions could have contribute to the early demise” (or similar).Δpks group vs the NC101 group considered a “small impact”? The 57 day survival is the NC101Δpks group but it is stated to the NC101 group and then median survival is stated as not able to be calculated but provided as 154 days previously. Please clarify for the reader.6) Why are half the mice surviving in the NC1017) The timing of the onset of colitis is unclear. Do the authors mean by 4 weeks of monocolonization or only following AOM?8) What was the timing of detection of the anal carcinoma?9) The mouse colon is well known to have a distal “smooth” part grossly and then the proximal half has a “feathered” gross structure. Please provide clarity on whether distal colon refers to the smooth section of the mouse colon or not. For example, in the enterotoxigenic B fragilis model of colon tumorigenesis, the transition between the smooth and feathered colon in the most severely affected animals is essentially a “hard stop” for further tumorigenesis . Is that is what is being described here?Δpks”. Was this true even when mice were harvested at a time point close to the time that mice were harvested in the original Arthur et al. paper ? The tumorigenesis results of any mice harvested near the timing of the original Arthur et al. paper should be commented on specifically. It seems possible that the time line in this model is crucial for differentiating the outcomes of NC101 and NC101Δpks.10) Please clarify the statement: “These observations were the same whether the mouse was mono-associated with NC101 or NC10111) What is the timing of the pictures in Figure 3—figure supplement 1?12) Same question as 10 above. Are there any mice analyzed at the time point of the harvests in the Arthur et al., paper?13) Suggested edit for clarity: change “14 weeks post-AOM treatment” to “14 weeks post-AOM treatment ”.14) Are the median scores reported correct? They have exactly the same numbers and statistical significance seems doubtful as reported. This requires either more explanation or correction.15) Suggest adding “more severe and/or progressive over time” to the sentence “The absolute scores were greater in this replication attempt compared to the original study, particularly for inflammation and invasion, which, combined with the survival and histopathological observations described above suggests that lesions were more severe in this replication attempt than in the original study.”16) Suggest adding after “AOM treatment” the following sentence: These results highlight the importance of experimental time lines in assessing differences in mouse models and between reports.” (or similar).17) “[…], had a natural tendency to have a reduced inflammatory response and tumor load compared to ones that died, then the data reported would be distorted”: The word “distorted” does not seem apt. Mouse models and mice within experiments can be remarkably variable including littermates, mice caged together etc. Isn‘t this the nature of mouse experiments and the point is that investigators need to be aware that inbred mice by no means provide clear “smoothing” of the data?18) “Additionally, under these conditions it is possible the products of excessive inflammatory responses  “This increased severity confounds the ability to detect differences […]”: Again, suggest changing wording to “increased severity over time confounds […]”20) Subsection “Meta-analyses of original and replicated effects”. Again, for clarity change “a common effect size was calculated for each effect from the original and replication studies” to “calculated for survival ”.Δpks were sequenced. Are these data submitted to GenBank? The accession numbers should be provided.21) Subsection “Genome sequencing data processing and assembly”. Both NC101 and NC101E. coli CFU”. What does “intestinal tissue feces” mean? Do the authors mean that intestinal luminal contents were removed from the colon at the time of mouse necropsy? Please clarify.22) Subsection “Determination of 23) Subsection “Statistical Analysis”. Suggest “reported in the original study in Supplemental […]”.24) Figure 1A,B should be revised to include much more data that would encapsulate the experiments and their contrast for the reader. Suggest adding numbers of mice for A,B experiments including number of males, females; mark the timing of mouse deaths with arrows; mark timing of onset of detection of colitis and tumors/invasive cancers ; add to legend what the cross lines added to the timeline represent . Ideally this figure would enable the reader to readily capture the contrasts between the studies and their timelines. Hopefully any needed information from Arthur et al. would be available to complete this.25) Figure 2 legend. (n=7 in 4 cages), correct?26) Figure 2—figure supplement 1. Add range of days/wks that sacrifices occurred.27) Figure 3—figure supplement 2 legend. Please add the timing of harvest of each mouse displayed in B,C, D and E, ideally on the figure. In Figure 3E, the herniation described is not really visible to the reader. Please provide a higher power inset. Similarly, please clarify Figure 3—figure supplement and Figure 3. Are these sections all from one mouse or different mice? What is the timing of the necropsies leading to these images? Sizing bars are missing from images A and C. In A, the “mucus lakes” should be marked and likely a higher power image of these provided. C appears to be at higher magnification than B. It would be best to show B and C at the same magnification.eLife. Your revised article has been favorably evaluated by Wendy Garrett (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Replication Study: Intestinal inflammation targets cancer-inducing activity of the microbiota" for further consideration at The manuscript has been improved and a highly responsive revision is again noted. However, there are some remaining issues that need to be addressed before acceptance, as outlined below:Two data concerns, both seem like typos:-/-Il10 mice mono-associated with E. coli NC101 or NC101𝛥pks”. Looking at Figure 2B, this reviewing editor thinks that NC101 and NC101Δpks are reversed. Namely, NC101(not NC101Δpks) median survival cannot be determined because more than 1/2 the mice were alive at 18 weeks following monoassociation.1) Subsection “Intestinal tumorigenesis and inflammation of germ-free -/-Il10 mice mono-associated with E. coli NC101 or NC101𝛥pks”. This reviewer thinks the authors are repeating the data although the language differs (lesions and macroscopic tumor burden). The authors note that a “statistically significant decrease in neoplastic lesions” reported in Arthur et al., but provide identical median scores. Please clarify.2) Subsection “Intestinal tumorigenesis and inflammation of germ-free  […] Essential revisions:As pointed out by all reviewers, the attempt at replication primarily revealed the technical aspects that must be controlled for in working with this mouse model in future experiments. A careful standardization of the protocol/disease course is required in individual laboratories before comparing different E. coli strains. Key features of the model to be verified before strain testing include low mortality rates, distinguishable individual tumors at the endpoint and additional controls including non-colonized AOM-injected-only control mice and mono-colonization-only and possibly AOM-untreated control mice. Revising the paper to focus on the technical limitations rather than that the replication attempt led to results differing from the original paper seems prudent. Ultimately, since the biology and kinetics of the Arthur et al., model were not faithfully replicated, these data are unable to address whether, under the conditions of the original paper, NC101 and ΔPKS NC101 differ in inflammation, invasion and neoplasia. This final point should be made clear to the readers of the paper.Thank you for sharing this critical information. We were unaware of the differences in experimental timing supplied by reviewer 4. As raised by reviewer 1 the original paper was not well described in terms of the timepoints. The experimental timing was based on the information in the original paper and described in the Registered Report with the mice to be sacrificed “18 weeks after last AOM injection”. This remained after informal review and feedback by the original authors during preparation of the Registered Report manuscript, peer review of the Registered Report, and post-publication peer review of the published Registered Report. This was also not raised in the other independent reviews of this Replication Study manuscript. One approach to mitigate the potential for misinterpreting complex study designs is to include a timeline diagram or flowchart as recommended by the ARRIVE Guidelines. We included these points in the revised manuscript.We also agree with the reviewer regarding the implications this has on the presentation and interpretation of the replication data. We have revised the figures, manuscript, and the Abstract to reflect the difference in experimental timing between the original study and this replication attempt. This includes removing the histopathological analysis from the meta-analysis and revising the survival meta-analysis to reflect the shared time frame between the two studies. We also revised the manuscript regarding the implications this has on the presentation and interpretation of the replication data.Reviewer #1:[…] Specific comments:1) Mortality seen on Figure 2A. These curves may represent two different types of mortalities (causes)- AOM induced mortality early on and then inflammation and/or tumor load induced mortality later.Early mortality in the Replication experiment is clearly higher than in Arthur et al. (>50% vs less than 30%).What is presented on Figure 2B may be very similar to the data on Supplementary Figure 10 by Arthur et al., , if mortality between day 0 and 10 is still attributed to the action of AOM. Then long time there is no mortality (days 20 to 80) in both Replication experiment and in Arthur et al. Then mice in Arthur et al., seemingly start to die, but they are immediately collected , and experiment stopped, so we do not know what happens to mortality. In Replication experiment past day 90 mortality starts but mice are euthanized according to symptoms or dying, so there is an impression of high mortality.We have revised this figure, the manuscript, and the Abstract to reflect the difference in experimental timing between the original study and this replication attempt in light of the information supplied by reviewer 4.2) One important discrepancy here is that in Arthur et al., 14 wk and 18 wk point mice were collected when they still were not dying, indicating that probably there is still a room for a difference in inflammation and tumor load/invasion.In Replication attempt, it seems from the Figure legend that mice were mostly collected and "declared dead" when they were already sick (right thing to do in terms of following IACUC regulation) but one could think that by the time PKS+ and ΔPKS mice are already equally very sick, tumor load and inflammation may be already too high and indistinguishable. Indeed, authors note that overall role of inflammation, tumor load and mortality in their experiment was higher than in initial experiment.We have revised the figures, the manuscript, and the Abstract to reflect the difference in experimental timing between the original study and this replication attempt in light of the information supplied by reviewer 4.3) Another thing noted during initial planning of experiments and review of Registered Report is that AOM quality may vary. Indeed, different lots of AOM may have different amount of "acting" AOM and sometimes it”s too much- more toxicity/mortality and more tumors in residual mice or poor decayed AOM- more toxicity/mortality and less tumors at the end. Seemingly, in Replication report higher mortality (presumably AOM induced) and higher tumor load is seen.We agree the observed toxicity is unexpected, especially since the dose and treatment schedule used is below the published toxic dose for AOM in mice. We have revised the manuscript and the Abstract to reflect the difference in experimental timing between the original study and this replication attempt in light of the information supplied by reviewer 4.4) Since AOM metabolism and potentially bacterial and liver metabolism may be involved, the diets used for germ free mice should be compared and that should be discussed.We agree that diets could be a factor and have included this in the revised manuscript. We also used the same diet as the original study  and have included this in the revised manuscript.5) Using Chi- values for tumor numbers in Replication study when tumors cannot be enumerated (why?) further complicates the interpretation.We revised the figure to better distinguish the tumors that could not be enumerated from the ones that could. This is included in the figure to illustrate the number that could not be quantified in both groups because of the coalescing nature of the lesions.6) Another important caveat which should be acknowledged also stems from high mortality. When mortality is that high, there may be a natural selection for mice with lower inflammatory response and lower tumor load- and the mice where potentially there was (could be) a difference have been kept for too long under these conditions of inflammation and AOM lot. This is not very scientific argument, I know, but after this Replication attempt is published, the field will need all of the possible scenarios/explanations discussed.We agree that the increased severity in the replication attempt complicates the interpretation of the data. Besides leading to information loss, if the mice that survived, and thus were quantified for inflammation and tumorigenesis, had a natural tendency to have a reduced inflammatory response and tumor load compared to ones that died, then the data reported would be distorted, limiting the opportunity to detect differences between the two groups. We have included this point in the revised manuscript.7) Inflammation is known to promote cancer. PKS+ bacteria is proposed to be more carcinogenic than PKSΔ. However, under the conditions of stronger inflammation (observed in Replication attempt), Inflammation arguably can become more important than PKS status. PKS is genotoxic, but so are the products of excessive inflammatory responses . One could argue that under stronger inflammation the presence or absence of PKS becomes less relevant as for its effect towards tumor progression. Therefore, comparison of this (Replication) and Arthur et al. studies with regard to tumor characteristics only makes sense if the same levels of inflammation are achieved.We have revised the figures, the manuscript, and the Abstract to reflect the difference in experimental timing between the original study and this replication attempt in light of the information supplied by reviewer 4.In my view, for these experiments the technical attempt to reproduce the results has been attempted but based on the confounded data it is difficult to judge whether important conceptual results are reproduced or not (i.e. whether PKS+ is superior at tumor progression).The only thing which can be concluded, in my view, is that during the Replication attempt, the team did not achieve the same levels of AOM toxicity/action, the same levels of inflammation and tumor load (more); and observed higher mortality. Under these circumstances it is still not clear whether PKS+ E. coli induces more tumors and more aggressive tumors; than ΔPKS controls.We have revised the figures, the manuscript, and the Abstract to reflect the difference in experimental timing between the original study and this replication attempt in light of the information supplied by reviewer 4.Reviewer #2:[…] Major points:-/- mice in order to evaluate the reproducibility of the experiments published by Arthur et al. For this comparison to be performed the kinetics of the model should be similar to what was reported by Arthur et al. In the present study mice mono-colonized with both E. coli strains display a much higher lethality and an exacerbated phenotype by histopathology as compared to the report by Arthur et al. A very low number of mice met the endpoint of the study. Because of these issues no conclusions can be drawn regarding the potential of E. coli NC101 vs NC101𝛥pks to induce intestinal inflammation and tumor formation in monocolonized GF Il10-/- mice.The major aim of the present study is to compare the potential of the E. coli strains NC101 and NC101𝛥pks in inducing intestinal inflammation and tumor formation in monocolonized GF Il10We have revised the figures, the manuscript, and the Abstract to reflect the difference in experimental timing between the original study and this replication attempt in light of the information supplied by reviewer 4.In the Abstract the authors first mention that "Mono-association […] resulted in similar levels of intestinal inflammation and tumorigenesis; whereas the original study reported decreased tumor multiplicity […]" and secondly, they mention that this replication study showed more severe histopathological observations and a much lower survival rate compared to the original study. This structure of the Abstract is misleading to the reader. The strong differences in the kinetics of the experiment compared to the original report and the lack of power of the current study to assess the reproducibility of the original findings should be mentioned first. The observations regarding tumor number and histopathological features could be discussed as indicative of a lack of difference in the context of a severely exacerbated disease course.We have revised the Abstract to reflect the difference in experimental timing between the original study and this replication attempt in light of the information supplied by reviewer 4.-/- mono-colonization model is characterized by important technical limitations that should be carefully controlled in future studies. A careful standardization of the protocol/disease course is required in individual laboratories before comparing different E. coli strains. This should lead to low mortality rates and distinguishable individual tumors at the endpoint. Including non-colonized AOM-injected-only control mice and mono-colonization-only, AOM-untreated control mice is essential for the correct interpretation of results in this model. Such factors should be considered for the experimental design of future studies.In my opinion, the major conclusion of this study is that the AOM/E. coli NC101 Il10We agree. Our findings clearly demonstrate the essential nature of clear and precise reporting of experimental details to ensure published research are accurately compared, reproduced, and interpreted. We have included these factors as considerations for the experimental design of future studies in the revised manuscript.Additional points:The comparison of the death rates between this study and Arthur et al. is vague in this part of the manuscript despite the fact that this is the major limitation of this replication attempt. The authors should clearly mention the percentages of mice meeting the endpoint in the two studies.We have revised the figures, the manuscript, and the abstract to reflect the difference in experimental timing between the original study and this replication attempt in light of the information supplied by reviewer 4. We have also moved the percentages of mice earlier in the text, when comparing death rates.Mortality was highest during AOM treatment independently of the bacterial strain used whereas mice started dying right after the first AOM injection. The interpretation of these observations is difficult because of the absence of AOM-only controls: lethality over the injection period may be an effect of AOM toxicity alone or of AOM in combination with the effect of E.coli. It may also be an effect of the injection process itself . This is an additional technical variable that should be mentioned in the Discussion.We agree and have included these factors as considerations for the experimental design of future studies in the revised manuscript.The authors mention that mortality during AOM injections was greater in mice colonized with NC101Δpks. Is this difference statistically significant? This should be mentioned and if significant further discussed.We have revised the figures, the manuscript, and the abstract to reflect the difference in experimental timing between the original study and this replication attempt in light of the information supplied by reviewer 4. Further discussion for the increased mortality during AOM injections was includedThe authors discuss sex-related differences which may confound the differences observed by Arthur et al. where 4 female and 8 male mice mono-associated with NC101 and 8 male mice mono-associated with NC101Δpks were analyzed. What is the result of these analyses if only male mice are considered? (n = 8 vs 8)?This is an interesting point. During preparation of the Registered Report we were informed of the ratio of female and male mice in their experiment; however, we did not receive the raw data from the authors that would allow us to conduct this exploratory analysis.The study is not powered enough to draw conclusions from the histopathological analyses because of the small number of mice resulting from an exacerbated disease course in the experiment.We agree we did not achieve the target number of 20 (instead reaching 10), which was based off the data from the 14 mice reported in the original study (at the 18 week timepoint). However, we have removed all analysis from the histopathological analysis in light of the information supplied by reviewer 4.Figure 2A: Log-rank test not shown in the legend.We included this exploratory test (of the entire timecourse) in the figure legend and manuscript in addition to the revised analysis of the comparable timecourse between the original study and this replication in light of the information supplied by reviewer 4.Reviewer #3:-/- mice were derived from the same germ-free colony used in the original study) , and the used AOM dose, 10 mg/kg, was the same as the original study . AOM administration  had resulted in a significantly higher mortality rate in this replication study compared with the original study. As cited by the authors , a previous study  showed that 33 mice were treated with 10 mg/kg of AOM, and "no premature loss of mice" was observed at "six months after the first AOM dose". This appears in line with the original study and indicates that 10 mg/kg of AOM should not cause substantial mortality rate; in contrast, AOM administration has resulted in a significantly higher mortality rate in this replication study, which could be due to the differences in AOM lots, mouse facilities, microbiota, etc. It appears reasonable that the difference in the effects of NC101 to NC101 Δpks on the intestinal inflammation and tumorigenesis, as reported in the original study, could not be replicated, with the more severe disease development in this replication study. However, in order to firmly evaluate the reproducibility of the original study, especially the effects of NC101 to NC101 Δpks on the intestinal inflammation and tumorigenesis, the experimental conditions (such as the administrated AOM dosage) could be adjusted in this replication study to get enough animals surviving through 18 weeks for the further analyses.[…] It appears that the authors of this replication study had tried the best to conduct the experiments under the conditions as close as possible to the original study. For instance, bacterial strains were shared by the Arthur Lab , germ-free Il10We have revised the figures, the manuscript, and the Abstract to reflect the difference in experimental timing between the original study and this replication attempt in light of the information supplied by reviewer 4.Reviewer #4:[…] Essential revisions:1) In both studies, colonization was established for 4 weeks, after which AOM was administered for 6 weeks. However, the current study evaluated survival and tumorigenesis 18 weeks after the last (6th) AOM injection, whereas the original 2012 study evaluated survival and tumorigenesis 18 weeks after colonization. This equates to 10 weeks longer in the current study, which is 50% greater time than in the original, and could explain why no difference in tumorigenesis was observed. The authors in fact indicate that it was difficult to detect differences in tumorigenesis because it had advanced so far and many mice did not survive to this time point. This substantial difference in experimental timing – 18 weeks (2012 study) and 28 weeks (current study) – is a major flaw in this replication study. This difference should be clearly stated in the Abstract and manuscript text. Further, I”m not sure if the meta-analysis in Figure 4 is appropriate with such differences in time frame between the two studies.Thank you for sharing this critical information. We were unaware of these differences in experimental timing. As raised by reviewer 1 the original paper was not well described in terms of the timepoints. The experimental timing was based on the information in the original paper and described in the Registered Report with the mice to be sacrificed “18 weeks after last AOM injection”. This remained after informal review and feedback by the original authors during preparation of the Registered Report manuscript, peer review of the Registered Report, and post-publication peer review of the published Registered Report. This was also not raised in the other independent reviews of this Replication Study manuscript. One approach to mitigate the potential for misinterpreting complex study designs is to include a timeline diagram or flowchart as recommended by the ARRIVE Guidelines. We included these points in the revised manuscript.We also agree with the reviewer regarding the implications this has on the presentation and interpretation of the replication data. We have revised the figures, manuscript, and the Abstract to reflect the difference in experimental timing between the original study and this replication attempt. This includes removing the histopathological analysis from the meta-analysis and revising the survival meta-analysis to reflect the shared time frame between the two studies.-/- studies. Perhaps the current authors injected mice with a very concentrated AOM solution? Survival curves from the original 2012 paper do not indicate this extent of toxicity. I recommend the authors amend their Abstract and manuscript text to indicate how different these results were between the current and former studies.2) Potential AOM toxicity: It is concerning that AOM treatment killed 30-70% of the mice, simply during the 6 week injection period. I am not aware of this extent of toxicity in AOM/DSS or AOM/Il10We agree the observed toxicity is unexpected, especially since the dose and treatment schedule used is below the published toxic dose for AOM in mice and, to our knowledge, is the same used in the original study. The cause of early death following AOM injection was confirmed to be acute liver failure, based on histologic evaluation. Inclusion of AOM-only controls could have indicated any increased susceptibility to AOM of the mice used in this study. We have also revised the Abstract to reflect the increased severity between the two studies during AOM treatment.3) Figure 2 figure legend mentions "animals where bacterial contamination was detected (7 out of 84) were censored". What does this mean? Does this mean that some animals housed in gnotobiotic (mono-associated) isolators became contaminated? This is highly concerning. It also raises the possibility that these mice were also infected with other non-bacterial microorganisms, such as virus or fungus. Any contamination could alter results. Potential contamination is only mentioned in this figure legend and does not appear to be mentioned in Results section or Materials and methods section. The authors must explain in greater detail what this "bacterial contamination" means – both to this reviewer and to the readers.We have revised the manuscript  to provide a clearer description of the contaminated mice. In brief, (1) the mice were in isocages, not isolators, with only a few isocages contaminated during the course of the experiment, and (2) the rest of the isocages remained gnotobiotic.The authors provided a highly responsive revision of the original paper. In re-reviewing Eaton, 2015 (study plan) and the original Arthur et al., paper (2012), we agree that the time line for the mouse experiments proposed in Eaton, 2015 was clear whereas the time line between bacterial inoculation, AOM administration and mouse harvest was hard to discern in the original paper. Besides the importance of clearer time line delineation in original manuscripts, this highlights flaws in the review of the original study plan, certainly a distributed responsibility in which several reviews failed to raise any questions.Thus, in the review of the current manuscript, there a number of places where the text should be modified to be clearer and more detailed as listed below.1) Introduction. The authors state incorrectly that in Arthur et al. a 100-fold increase in E. coli NC101 was detected in the lumen of Il10 KO mice relative to WT controls. This is Figure 1I in Arthur et al. In this experiment, conducted at 20 weeks after transfer to SPF conditions, only the presence of E. coli was examined, not NC101 which would have required specific testing for pks or colibactin. NC101 is referred to but, not specifically tested for at this juncture in the Arthur et al., paper.Thank you for raising this error. We have revised the manuscript to remove this statement.-/- mice mono-associated with E. coli NC101 or NC101𝛥pks”. It was difficult to find wording in the Arthur et al. paper that clearly delineated whether harvest occurred 18 weeks from monoassociation vs 18 weeks post-AOM (wording was “14 and 18 weeks with AOM”) and better definition was missed by several reviews of the Eaton et al. plan including, even perhaps the original authors, as such the following wording change is suggested: “based on methods derived from the original study and not corrected on review of Eaton et al. As this may represent a more accurate presentation of what occurred.2) Subsection “Intestinal tumorigenesis and inflammation of germ-free Il10We agree and have revised this sentence as suggested.-/- mice mono-associated with E. coli NC101 or NC101𝛥pks”. Need to insert here the starting N of mice  since the reader can’t interpret the mouse survival numbers without this information.3) Subsection “Intestinal tumorigenesis and inflammation of germ-free Il10We agree and have included this in the revised manuscript.4) Please add the range of days of survival to augment the median survival stated.We have included the range of survival times for each group.5) Please expand “suggesting bacterial load was not a factor in the survival differences” as follows: “suggesting while bacterial load was not a factor in the survival differences, host:bacterial interactions could have contribute to the early demise” (or similar).While the interpretation that bacterial load was not a factor, it is not clear that host:bacterial interactions necessarily contributed. The early death rate in response to AOM was not explained in this study and thus we feel it is not appropriate to speculate.6) There to be an error. Why are half the mice surviving in the NC101Δpks group vs the NC101 group considered a “small impact”? The 57 day survival is the NC101Δpks group but it is stated to the NC101 group and then median survival is stated as not able to be calculated but provided as 154 days previously. Please clarify for the reader.This analysis was to compare the replication results to the original study. In order to do that we treated 18 weeks after mono-association as the study end point . We have revised the manuscript to better clarify what these results represent. We have also removed the word “small” as this was indeed an error.7) The timing of the onset of colitis is unclear. Do the authors mean by 4 weeks of monocolonization or only following AOM?We have revised the manuscript to reflect the timing of the onset of colitis. Specifically, the first mouse was at 5 weeks after monocolonization.8) What was the timing of detection of the anal carcinoma?We have included the timing of the anal carcinoma in the revised manuscript. Specifically, this was observed between 19 and 27 weeks after monocolonization.9) The mouse colon is well known to have a distal “smooth” part grossly and then the proximal half has a “feathered” gross structure. Please provide clarity on whether distal colon refers to the smooth section of the mouse colon or not. For example, in the enterotoxigenic B fragilis model of colon tumorigenesis, the transition between the smooth and feathered colon in the most severely affected animals is essentially a “hard stop” for further tumorigenesis . Is that what is being described here?We have revised this section to better clarify the observations. The most extensive tumors reached approximately mid-colon. The gross appearance of the proximal mucosa was not recorded.10) Please clarify the statement: “These observations were the same whether the mouse was mono-associated with NC101 or NC101Δpks”. Was this true even when mice were harvested at a time point close to the time that mice were harvested in the original Arthur et al. paper ? The tumorigenesis results of any mice harvested near the timing of the original Arthur et al. paper should be commented on specifically. It seems possible that the time line in this model is crucial for differentiating the outcomes of NC101 and NC101Δpks.We have revised this section to clarify the range these observations were made. We also included in a previous paragraph the observations of the mice that were harvested at times close to the original study.11) What is the timing of the pictures in Figure 3—figure supplement 1?This is from a mouse that was mono-associated with NC101 and died 13 weeks after AOM treatment. The timing is described in the figure legend.12) Are there any mice analyzed at the time point of the harvests in the Arthur et al., paper?We included the available information in the paragraph included in response to point 10 above.13) Suggested edit for clarity: change “14 weeks post-AOM treatment” to “14 weeks post-AOM treatment ”.We agree and included this in the revised manuscript.-/- mice mono-associated with E. coli NC101 or NC101𝛥pks”. Are these scores correct? They have exactly the same numbers and statistical significance seems doubtful as reported. This requires either more explanation or correction.14) Subsection “Intestinal tumorigenesis and inflammation of germ-free Il10t test), which is not appropriate since the mean cannot be defined . That is, while a number is used for the scoring it represents non-numeric concepts like “severe or high grade dysplasia characterized as adenoma, restricted to the mucosa”. We conducted a non-parametric test (i.e. Mann Whitney test) on the original data which gave similar results. We included this additional explanation in the revised manuscript.These are the median scores reported in the original study. The original study analyzed the ordinal scoring data as interval measurements (by 15) Suggest adding “more severe and/or progressive over time” to the sentence “The absolute scores were greater in this replication attempt compared to the original study, particularly for inflammation and invasion, which, combined with the survival and histopathological observations described above suggests that lesions were more severe in this replication attempt than in the original study.”We agree and included this in the revised manuscript. We have also revised this section to better clarify the observations in light of the differences in methodology between the two studies.-/- mice mono-associated with E. coli NC101 or NC101𝛥pks”. Suggest adding after “AOM treatment” the following sentence: These results highlight the importance of experimental time lines in assessing differences in mouse models and between reports.” (or similar).16) Subsection “Intestinal tumorigenesis and inflammation of germ-free Il10pks. Additionally, we have included a sentence that methods should be clearly described and published to facilitate reproducibility.We have revised this section to highlight that it is possible that the experimental timing in this mouse model is crucial for differentiating the outcomes of NC101 and NC101Δ17) “[…], had a natural tendency to have a reduced inflammatory response and tumor load compared to ones that died, then the data reported would be distorted”: The word “distorted” does not seem apt. Mouse models and mice within experiments can be remarkably variable including littermates, mice caged together etc. Isn’t this the nature of mouse experiments and the point is that investigators need to be aware that inbred mice by no means provide clear “smoothing” of the data?We removed this sentence and revised this section to better clarify the observations in light of the differences in methodology between the two studies.18) “Additionally, under these conditions it is possible the products of excessive inflammatory responses  “This increased severity confounds the ability to detect differences […]”: Again, suggest changing wording to “increased severity over time confounds […]”We removed this sentence and revised this section to better clarify the observations in light of the differences in methodology between the two studies.20) Subsection “Meta-analyses of original and replicated effects”. Again, for clarity change “a common effect size was calculated for each effect from the original and replication studies” to “calculated for survival ”.We agree and included this in the revised manuscript.21) Subsection “Genome sequencing data processing and assembly”. Both NC101 and NC101Δpks were sequenced. Are these data submitted to GenBank? The accession numbers should be provided.We have included the accession numbers in the revised manuscript.22) Subsection “Determination of E. coli CFU”. What does “intestinal tissue feces” mean? Do the authors mean that intestinal luminal contents were removed from the colon at the time of mouse necropsy? Please clarify.This was a typographic error. “Intestinal tissue” has been deleted.23) Subsection “Statistical Analysis”. Suggest “reported in the original study in Supplemental […]”.We agree and included this in the revised manuscript.24) Figure 1A,B should be revised to include much more data that would encapsulate the experiments and their contrast for the reader. Suggest adding numbers of mice for A,B experiments including number of males, females; mark the timing of mouse deaths with arrows; mark timing of onset of detection of colitis and tumors/invasive cancers ; add to legend what the cross lines added to the timeline represent . Ideally this figure would enable the reader to readily capture the contrasts between the studies and their timelines. Hopefully any needed information from Arthur et al. would be available to complete this.1We have revised Figure 2A,B to include more information as suggested and included what information was available from Arthur et al. The timing of mouse deaths is already indicated in the Kaplan-Meier plot in the vertical lines, thus we did not include additional marks.25) Figure 2 legend. (n=7 in 4 cages), correct?There were only 3 cages. We have revised the figure legend to accurate reflect this in addition to the number of mice (n=7).26) Figure 2—figure supplement 1. Add range of days/wks that sacrifices occurred.We have included the range of sacrifices.27) Figure 3—figure supplement 2 legend. Please add the timing of harvest of each mouse displayed in B,C, D and E, ideally on the figure. In Figure 3E, the herniation described is not really visible to the reader. Please provide a higher power inset. Similarly, please clarify Figure 3—figure supplement 3 and Figure 3. Are these sections all from one mouse or different mice? What is the timing of the necropsies leading to these images? Sizing bars are missing from images A and C. In A, the “mucus lakes” should be marked and likely a higher power image of these provided. C appears to be at higher magnification than B. It would be best to show B and C at the same magnification.The time of euthanasia has been added to the figure legends. The herniated gland in Figure 3—figure supplement 2E has been circled to clarify it. Size bars have been added to Figure 3—figure supplement 3. Figure 3—figure supplement B,C are at different magnifications because they demonstrate different distributions of lesions. Parts A and C are from the same mouse, while part B is from a different mouse.The manuscript has been improved and a highly responsive revision is again noted. However, there are some remaining issues that need to be addressed before acceptance, as outlined below:Two data concerns--both seem like typos:-/- mice mono-associated with E. 124 coli NC101 or NC101𝛥pks”. Looking at Figure 2B, this reviewing Editor thinks that NC101 and NC101Δpks are reversed. Namely, NC101(not NC101Δpks) median survival cannot be determined because more than 1/2 the mice were alive at 18 weeks following monoassociation.1) Subsection “Intestinal tumorigenesis and inflammation of germ-free Il10Thank you for catching this error. We have corrected it in the revised manuscript.-/- mice mono-associated with E. coliE. coli NC101 or NC101𝛥pks”. This reviewer thinks the authors are repeating the data although the language differs (lesions and macroscopic tumor burden). The authors note that a “statistically significant decrease in neoplastic lesions” reported in Arthur et al., but provide identical median scores. Please clarify.2) Subsection “Intestinal tumorigenesis and inflammation of germ-free Il10Thank you for raising these. One reference was to the number of macroscopic tumors, while the other referred to the scoring of tissues for neoplasia. We have revised the latter sentence from “neoplastic lesions” to “neoplasia scores” to avoid any confusion and describe the invasion and inflammation scores in the same manner.Δpks are identical (both are 4). While a t-test was not appropriate for these data, a U-test gave the same results, which we report. The statistical difference is because of the spread of the data, despite identical medians. We have included the range for each measure to help clarify this point.The original median scores for neoplasia, invasion, and inflammation are accurately reported and the reviewer is correct that the median scores for NC101 and NC101


Naja nigricollis, oxidises oxyhaemoglobin (Fe2+) into methaemoglobin (Fe3+) in a time- and concentration-dependent manner that is unparalleled within the 47 viper and elapid venoms evaluated. The treatment of venom with a reducing agent, dithiothreitol (DTT) is observed to potentiate this effect at higher concentrations, and the use of denatured venom demonstrates that this effect is dependent upon the heat-sensitive proteinaceous elements of the venom. Together, our results suggest that Naja nigricollis venom appears to promote methaemoglobin production to a degree that is rare within the Elapidae family, and this activity appears to be independent of proteolytic activities of venom components on haemoglobin.Snakebite envenomation is an affliction currently estimated to be killing upwards of 100,000 people annually. Snakebite is associated with a diverse pathophysiology due to the magnitude of variation in venom composition that is observed worldwide. The haemolytic  actions of snake venoms are well documented, although the direct impact of venoms on haemoglobin is not fully understood. Here we report on the varied ability of a multitude of snake venoms to oxidise haemoglobin into methaemoglobin. Moreover, our results demonstrate that the venom of an elapid, the black necked spitting cobra,  Snakebite envenomation is considered to be a major neglected tropical disease. It is estimated to affect nearly five million people and result in approximately 100,000 deaths annually ,3. DespiA range of biological activities have been found to be induced by snake venom components, with many of these proteins targeting the blood, haemostasis and cardiovascular system ,7. SerinIn addition to natural intra- or extravascular haemolysis (approximately 10% of RBCs will lyse as a consequence of wear and tear) and direct lysis by venom components, haemolysis also takes place as a result of the venom-induced local tissue damage which produces reactive oxygen (ROS) and nitrogen species that contribute to oxidative damage and further lysis of cells . The rel2 (or dissolved HCO3−) to the lungs; they are entirely dependent upon the oxygen transport protein, Hb. This protein relies on a prosthetic ferrous (Fe2+) haem group, the ferric oxidation of which renders the protein (termed as methaemoglobin (MetHb)) unable to transport oxygen (Fe3+), a process which has already been observed in specific snake venoms  and then incubated with ovine Hb. The presence of DTT failed to prevent the oxidation of Hb, and indeed increased the production of MetHb at the highest concentration (5 mM), although at lower concentrations this increase was not significant  venom was found to have a greater ability to produce MetHb than two other Indian vipers (Echis carinatus and Daboia russelii). Those results are inconsistent with the findings from this study where many of the viper venoms including Echis carinatus had more pronounced effects than Naja naja venom. Although the reasons for these discrepancies are not entirely clear, they have used human whole blood in their experiments, whereas we have used ovine Hb. Therefore, their experiments may relate more to the ability of venoms to lyse RBCs (as Hb would be much more accessible after lysis) than to directly oxidise Hb. We can postulate that single or multiple venom components may act either directly on Hb or potentially on MetHb reductase to prevent the equilibrium found under healthy natural conditions. A schematic diagram of our working hypothetical model of the impact of venom components on the production of MetHb is shown in In a previous study , Naja naCrotalus molossus nigrescens venom reported that high concentrations of this venom cause some level of proteolytic degradation to Hb [2O2 which is produced by venom LAAO [2O2 then reacts with the ferrous Hb producing further reactive species via the Fenton reaction [N. nigricollis is a snake with potently cytotoxic venom that lacks LAAOs and is composed largely of three-finger toxins (73.3%) and PLA2 (21.9%) [N. nigricollis venom supports the theory that the phospholipase activity could be indirectly causing this effect as a result of its oxidative product, arachidonic acid. However, members of the genus Agkistrodon screened for their Hb oxidising ability  venom, and it may be possible that similar peroxidation takes place as a result of N. nigricollis venom.A previous study using on to Hb . The samnom LAAO . It may reaction . However (21.9%) . These p ability  were fouis venom , componeis venom , releasiis venom . Given txic, ROS . The incN. nigricollis venom demonstrates that the components of this venom are able to promote the oxidation of Hb. The increase in MetHb production observed over 16 h and at higher venom concentrations suggests that this effect may increase over time in snakebite victims, and is dependent on the volume of venom injected by the snake. The increase in MetHb production observed with the venom that was treated with the highest concentration of DTT suggests that this effect is actually improved by the addition of a redox reagent. However, at lower concentrations of DTT, this increase was not observed. We originally hypothesised that the addition of a redox reagent such as DTT would prevent the production of ROS, and consequently MetHb by taking the place of antioxidants such as glutathione found naturally in the plasma [N. nigricollis venom on Hb and to further scrutinise the mechanism of action of this venom on MetHb production.The time- A,B and ce plasma . The thiVenom-induced neurological and coagulation pathologies have long been the two major areas of interest for scientists to investigate snakebite pathophysiology due to their prominence as the most immediately life-threatening aspects of a snakebite. However, the morbidity is gaining interest recently as estimates of as many as 15,000 people annually suffering amputations as a result of snakebites in sub-Saharan Africa alone . Hence, N. nigricollis venom for the production of MetHb. This effect is likely to be a result of proteins present in the venom, although further fractionation and characterisation is required to identify the exact proteins causing these changes. Moreover, most of the experiments performed in this study used ovine Hb due to availability. However, future experiments will be performed using human RBCs and Hb. The clinical significance of venoms on MetHb production and its contribution to envenomation will also be determined in the future. The work highlighting co-administration of melatonin with ASV is worth pursuing further [In conclusion, we have demonstrated that a wide variety of venoms possess Hb-modifying activity among a range of viper and elapid snakes. From the venoms tested, we have observed a significantly higher activity for  further , and the further . AlthougCV) of whole snake venoms was from Venomtech Limited  and ovine blood was obtained from Envigo (UK) Ltd. . Clear 96- and 384-well plates were purchased from Greiner , and DTT, purified bovine Hb, and all other chemicals were from Sigma Aldrich The cardiovascular targeted-venom discovery array , and the clear supernatant was collected and used in further assays as ovine Hb. Although this supernatant is likely to have other components from RBCs, the presence of a prominent Hb band was confirmed by SDS-PAGE prior to using this as a Hb source in further functional assays.CV array were diluted in 1× PBS  (0.2 mg/mL) and mixed with an equal volume of ovine Hb (diluted 1:2 in 1× PBS) with a final assay volume of 100 µL in a 96-well micro titre plate. These samples were then incubated at 37 °C and the absorbance at 500 nm and 630 nm was measured at 16 h by spectrofluorimetry .The whole snake venoms from the T-VDAN. nigricollis venom (at 10 mg/mL or a range of concentrations between 1 mg/mL and 0.007 mg/mL) or PBS (a negative control) in a 384-well plate. Plates were incubated at 37 °C in a plate reader  and the absorbance spectrum between 400 nm and 700 nm was measured at 2-h intervals over 16 h. After this time, maximum Hb oxidisation appeared to be reached, and therefore, this time point was chosen for other experiments in this study. Moreover, to determine the impact of heat and a reducing agent, venom was heat-treated (65 °C or 95 °C in a dry bath) or treated with a reducing agent, DTT  prior to using them in assays. The temperatures (65 °C or 95 °C) were determined based on our previous experience in denaturing venom proteins and using them in functional assays. DTT was specifically selected as this is a less toxic and volatile compared to β-mercaptoethanol; moreover, DTT prevents the reoxidisation of disulfide bonds in proteins.Ovine Hb diluted (1:2) in 1× PBS was incubated with 2O).The SDS-PAGE analysis was performed according to standard procedures as described previously . The samp-Values were calculated using one-way or two-way ANOVA followed by Tukey’s post-hoc multiple comparisons test.All statistical analyses were performed using GraphPad Prism 7.