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. 2023 Jun 9;18(6):e0287145. doi: 10.1371/journal.pone.0287145

Effects of fenbendazole on fecal microbiome in BPH/5 mice, a model of hypertension and obesity, a brief report

Kalie F Beckers 1, Christopher J Schulz 2, Chin-Chi Liu 1, Elise D Barras 1, Gary W Childers 2, Rhett W Stout 1, Jenny L Sones 1,3,*
Editor: Christopher Staley4
PMCID: PMC10256194  PMID: 37294797

Abstract

Fenbendazole (FBZ) is a common antiparasitic treatment used in research rodent colonies for biosecurity purposes. The effect of this compound has been studied in C57 mice, but never before in a strain of mice that has co-morbidities, such as the blood pressure high (BPH)/5. The BPH/5 mouse is an inbred genetic model of hypertension. While both male and female BPH/5 have high blood pressure, there is a metabolic sexual dimorphism with females displaying key features of obesity. The obese gut microbiome has been linked to hypertension. Therefore, we hypothesized that fenbendazole treatment will alter the gut microbiome in hypertensive mice in a sex dependent manner. To test the influence of FBZ on the BPH/5 gut microbiota, fecal samples were collected pre- and post-treatment from adult BPH/5 mice (males and non-pregnant females). The mice were treated with fenbendazole impregnated feed for five weeks. Post-treatment feces were collected at the end of the treatment period and DNA was extracted, and the V4 region of 16S rRNA was amplified and sequenced using the Illumina MiSeq system. The purpose was to analyze the fecal microbiome before and after FBZ treatment, the results demonstrate changes with treatment in a sex dependent manner. More specifically, differences in community composition were detected in BPH/5 non-pregnant female and males using Bray-Curtis dissimilarity as a measure of beta-diversity (treatment p = 0.002). The ratio of Firmicutes to Bacteroidetes, which has been identified in cases of obesity, was not altered. Yet, Verrucomicrobia was increased in BPH/5 males and females post-treatment and was significantly different by sex (treatment p = 5.85e-05, sex p = 0.0151, and interaction p = 0.045), while Actinobacteria was decreased in the post-treatment mice (treatment p = 0.00017, sex p = 0.5, interaction p = 0.2). These results are indicative of gut dysbiosis compared to pre-treatment controls. Lactobacillus was decreased with FBZ treatment in BPH/5 females only. In conclusion, fenbendazole does alter the gut microbial communities, most notable in the male rather than female BPH/5 mouse. This provides evidence that caution should be taken when providing any gut altering treatments before or during mouse experiments.

Introduction

Fenbendazole (FBZ) is a broad spectrum benzimidazole anthelmintic that is commonly used in laboratory animals for the treatment of pinworms [1]. The drug is regarded as safe, including use during pregnancy, and has minimal side effects [1]. FBZ impregnated feed is the current treatment of choice to eliminate rodent internal parasites. While poorly absorbed, bioavailability is enhanced when taken with a meal [2]. Benzimidazoles act by altering microtubule function through inhibition of microtubule polymerization and binding to B-tubulin [3]. Benzimidazoles have been shown to have adulticidal, larvicidal, and ovicidal properties [4,5]. Studies in rodents have indicated multiple physiologic effects of FBZ, including changes in immune parameters [6–8], but more research is needed to evaluate the effects of FBZ on the gut microbiome in a sex dependent manner in mice with co-morbidities, which are phenotypic hallmarks of genetic mouse models.

The treatment used in this study was FBZ impregnated Purina 5001 chow fed ad libitum for five weeks. The treatment duration considers the lifecycle of the parasites as well as the persistence of the parasite eggs in the environment. Pinworms (order Oxyurina) are one of the most common parasites found in modern rodent facilities [9–11]. Typically in mice, pinworm infestations are subclinical, but heavy burdens can lead to rectal prolapse, enteritis, and intussusception, especially in strains with co-morbidities [12–14]. Decreased weight gain, diminished growth rates, increased caloric demands, and overall nutritional status can be compromised [12,15,16]. Parasite load can also be influenced by the animals age, weight, strain/stock and sex [10,11,13]. As a result, infections may influence experimental outcomes.

The ideal gut microbiome is rich and diverse. Changes in the gut microbiome are referred to as a dysbiosis. Dysbiosis can lead to adverse health outcomes. This study aims to evaluate the changes in the gut microbiome pre- and post-five-week treatment of FBZ in the BPH/5 (blood pressure high subline 5) mouse model. The BPH/5 mice demonstrate cardiometabolic disease spontaneously [17,18]. This mouse model has been used to study preeclampsia (PE) [18–22]. Because this mouse spontaneously develops PE, increased blood pressure during pregnancy, we can investigate pre-pregnancy risk factors. The BPH/5 females have elevations in blood pressure and obesity compared to C57 females before pregnancy [23]. The BPH/5 males demonstrate increased blood pressure without obesity compared to C57 males unlike the BPH/5 females [24]. The overarching hypothesis is that FBZ treatment will alter the gut microbiome in a sex dependent manner in BPH/5 mice. This study will compare the microbial communities of the BPH/5 male and female pre- and post-treatment to evaluate the effect of FBZ on the gut microbiome on a mouse strain with coupled comorbidities.

Materials and methods

Animal experiments

This is a cross-over study evaluating the effects of fenbendazole (FBZ) on the gut microbiome in a sex dependent manner. Adult (age range: 8 weeks-6 months of age) female and male BPH/5 mice were used in this study. BPH/5 mice were a gift from Dr. Robin Davisson, Cornell University. They are maintained as an in-house colony at Louisiana State University. Mice were housed in Micro-Isolator™ cages (Lab Products, LLC. Aberdeen, Maryland, USA) on individually ventilated cage racks (Lab Products, LLC. Aberdeen, Maryland, USA). The cages are placed in a temperature- and humidity-controlled facility, maintained on a 12-hour light/dark cycle, and fed standard mouse chow (LabDiet 5001 Laboratory Rodent Diet, LabDiet, St. Louis, MO, USA) with water available ad libitum. The mice were co-housed and randomly selected for sampling. Two female mice were lost before follow up and one male pre-treatment sample had insufficient fecal material collected. Two groups of mice were used: 1) BPH/5 pre- and post- treatment males (n = 9 and n = 10, respectively) and 2) BPH/5 pre- and post- treatment non-pregnant females (n = 12 and n = 10, respectively). The pre-treatment mouse served as its own control in this study. All procedures were approved by Louisiana State University’s Institutional Animal Care and Use Committee.

Treatment

Mice were fed FBZ impregnated feed (Mod LabDiet 5001 w/150ppm Fenbendazole, LabDiet, St. Lous, MO, USA) ad libitum for five weeks. Complete cage changes occurred every two weeks in which feed was completely changed out during this time. Feed was topped off as needed in between cage changes. Cages (including the box, lid, cage card holder, feed hopper, shelter, water bottle, and water bottle lids) were cleaned via a cagewasher (NorthStar Model R630, BetterBuilt, Delta, BC, Canada) every two weeks. Cages are washed for 25 minutes using Uri-Solve (Pharmacal Research Laboratories, Inc. Waterbury, CT, USA) and Clout (Pharmacal Research Laboratories, Inc. Waterbury, CT, USA) cleaning agents with a final rinse temperature of 180℉. Personal Protective Equipment (PPE) was donned and doffed prior to entering and exiting the room, respectively. PPE included shoe covers, a disposable gown, nitrile gloves, and a hair net.

Samples collection

Fecal samples were collected pre-treatment and at the end of the five weeks post treatment. To collect feces, mice were placed individually in sterile empty cages and allowed to defecate voluntarily. Samples were placed in sterile tubes and stored at –80°C until further analysis.

DNA sequencing

Microbial DNA was extracted from fecal samples using the Qiagen DNeasy PowerSoil extraction kits (Qiagen, USA) according to manufacturer’s protocol, with the initial step of the protocol beginning with vertexing the sample with beads to break up the material. The V4 variable region of the 16S rRNA gene was amplified with PCR primers 515f/806r [25] in a 30 cycle PCR using the DreamTaq Hot Start PCR Master Mix Kit (Thermoscieniftic, Waltham, MA). PCR was performed in 20 μl vol and included: 2 μl (7.5 μM concn) of forward and reverse primers, 12.5 μl of Hot Start Taq 2X Master Mix (New England BioLabs Inc., Ipswich, MA., USA), 3.5 μl of deionized water, and 2 μl of sample DNA. Thermal cycle conditions were 95˚C for 3 min for initial denaturing step, followed by 30 cycles of 95˚C for 30 s, 50˚C for 1 min, and 72˚C for 1 min. PCR products were checked on a 2% agarose gel for correct product size formation (approx 350 bp). Michigan State University Genomics Core performed library preparation prior to Illumina MiSeq sequencing following the manufacturer’s guidelines [25,26]. Reagent controls using certified DNA free water were run through library preparation and PCR and did not generate libraries. For quality control, samples submitted for sequencing included a random blank sample of technical replicates.

Bioinformatics

Initial quality screening, demultiplexing, amplicon sequence variant (ASV) inference and chimera removal were performed using the DADA2 package [27]. ASVs were classified using the Silva Release 132 16S rRNA database [28,29]. Microbial community analysis (Alpha and Beta Diversity) was performed using the vegan R package [30]. Shannon diversity index was used to analyze alpha diversity, while Bray-Curtis dissimilarity was used to examine beta diversity. Permutational multivariate analysis of variance (PERMANOVA) [31] was performed using vegan package Adonis function. Statistical analysis was performed using GraphPad Prism Version 9.5.0 for Windows (GraphPad Software Inc., San Diego, CA). To determine differentially abundant ASVs, the ASV table was first trimmed to only include ASVs with an abundance of two or more ASVs across all samples. Two-way ANOVAs were performed on the trimmed ASV table using centered log ratio transformed abundances with time, sex, time x sex as the fixed effects and each mouse as the random effect [32]. Shannon diversity index against with treatment, gender and their interactions. ANOVA p-values were corrected using the False Discovery Rate (FDR) method of Benjamini and Hochberg [33]. All figures were presented as means± SEM. P values and FDR-corrected p values <0.05 were considered significant. All raw sequence reads and corresponding metadata can be found on the SRA website accession number PRJNA851094.

Results

Changes at the phylum level in BPH/5 mice with Fenbendazole treatment

Of the 40 samples processed and sequenced, all achieved a threshold above 10,000 reads with an average of 76,524 per sample after quality filtering. A bar graph was used to visualize the relative abundance at the phylum level within each group (Fig 1). The top phyla consisted of Firmicutes, Bacteroidetes, Epsilonbacteraeota, and Proteobacteria. The BPH/5 males had an increase relative abundance in Firmicutes from 39.44% to 49.67% with a decrease in Bacteroidetes of 48.40% to 36.81% post-treatment (Fig 2A). Epsilonbacteraeota remained similar with 7.44% and 7.96% pre- and post-treatment, similarly Proteobacteria and Cyanobacteria did not vary with 1.74% and 1.86%, and 2.12% and 1.86% pre and post, respectively (Fig 2A). Tenericutes decreased from 0.053% to undetected and Verrucomicrobia increased from undetected to 1.64% (Fig 2A). The BPH/5 females showed more similar relative abundances pre- and post-treatment (Fig 2B). A slight decrease in Firmicutes from 47.11% to 44.7%, Bacteroidetes remained similar 41.17% and 39.51%, slight increase in Epsilonbacteraeota 6.61% to 10.65%, Proteobacteria remained unchanged 2.55% and 2.51% (Fig 2B). Cyanobacteria was not affected 2.17% and 2.21%, Verrucomicrobia nor Tenericutes were either, 0.00% and 0.17%, 0.01% and 0.00%, respectively (Fig 2B). The Firmicutes to Bacteroidetes ratio, which is an indicator of obesity in humans [34], was not significantly different between groups (Fig 2C). In addition, the centered log ratios (clr, log base 2) of the different phyla were assessed to standardize the abundances of the microbes. Verrucomicrobia increased post-treatment and had significant differences between sex and interaction between sex and treatment (treatment p = 5.85e-05, sex p = 0.0151, and interaction p = 0.045), (Fig 2D). This demonstrated that the treatment had an effect in a sex dependent manner. Actinobacteria significantly decreased post-treatment but did not have differences by sex or interaction (treatment p = 0.00017, sex p = 0.5, interaction p = 0.2). (Fig 2E).

Fig 1. Bar graph of the relative abundance at the phyla level of BPH5 mice with fenbendazole treatment.

Fig 1

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Fig 2. Differences at the phyla level of BPH5 male and females pre and post fenbendazole treatment.

Fig 2

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A). Relative abundance at the phyla level of the BPH5 male pre and post-treatment. B). Relative abundance at the phyla level of the BPH5 female pre and post-treatment. C). Ratio of Firmicutes to Bacteroidetes in BPH/5 male and females. D). Changes in Verrucomicrobia pre and post fenbendazole treatment in male and female BPH/5 mice. E). Changes in Actinobacteria pre and post fenbendazole treatment in male and female BPH/5 mice.

Microbial community composition of the BPH/5 mouse with Fenbendazole treatment

To assess changes in gut microbial diversity with treatment Shannon’s index was assessed. Diversity significantly decreased after FBZ treatment, but no difference was observed between sex or interaction between treatment and sex (treatment p = 0.02, sex p = 0.19, interaction p = 0.17) (Fig 3A). Since observed diversity can be affected by sampling effort, i.e., sequencing depth, samples were rarefied to 5000 observations per sample randomly with 1000 trials to assess the significance of treatment on Shannon’s index. Treatment was significant (p < 0.05) for all rarefied samplings with a range of 0.005 to 0.014. To determine if community composition was different between treatments (beta diversity), a Bray-Curtis dissimilarity matrix was calculated from the relative abundances of ASVs in samples. A principal coordinate analysis (PCoA) plot was created using the Bray-Curtis dissimilarity (Fig 3B). Separation between pre- and post- treatment samples was observed along the first two principal coordinates which accounted for 19.33% and 13.12% of the variance between the samples. Treatment had a significant effect on community composition using PERMANOVA of the Bray-Curtis dissimilarity matrix, with both BPH/5 female and male microbial communities differing post-treatment but not different by sex or interaction (treatment p = 0.002, sex p = 0.3, interaction p = 0.05) (Fig 3B).

Fig 3.

Fig 3

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A.) Alpha diversity of BPH5 mice treated with Fenbendazole B.) Principal coordinate analysis of Bray-Curtis dissimilarity matrix of BPH5 mice treated with Fenbendazole.

Changes at the ASV level with Fenbendazole treatment and phenotypic outcomes

Looking further there were changes at the ASV level using the centered log ratios (clr, log base 2) of the relative abundance mentioned above, the BPH/5 males had a relative decrease in Lachnoclostridium spp, Lachnospiraceae spp, Odoribacter spp, Clostridiales family Xiii ssp, Alistipes spp, and Muribaculceae ssp (Fig 4A). While in the BPH/5 females, Lactobacillus spp, Ruminococcus_1 ssp, and Muribaculaceae spp were all found to be decreased with FBZ treatment (Fig 4B). Since the phylum Verrucomicrobia was found to be significantly different by treatment, sex, and interaction it was investigated further. Akkermansia was the found at the genus level of the phyla Verrucomicrobia, Akkermansia (Verrucomicorbia) was significantly increased in males post treatment and was greater in males compared to females (Fig 2). Within the phylum Actinobacteria which was significantly reduced with treatment in both sexes, Enterorhabdus was the major genus that contributed to these changes.

Fig 4.

Fig 4

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Top 6 changes at ASV level in a sex dependent manner A). Changes found with fenbendazole treatment at the ASV level in BPH5 male. B). Changes found with fenbendazole treatment at the ASV level in BPH5 female.

Discussion

Routine veterinary care in mouse vivariums is necessary for colony maintenance. Herein we discovered that FBZ does alter the gut microbial communities when analyzing alpha and beta-diversity in adult BPH/5 females and males. The most notable changes were seen in the BPH/5 males where the change in diversity is seen at the phylum level. It has been well documented that FBZ treatment has no effect on reproduction or food intake in rodents [35]. Therefore, even though FBZ is safe to use it could be altering the gut microbiome and changing experimental results in animals with co-morbidities.

A study by Korte et al, evaluated the gut microbiome on C57 female mice using FBZ impregnated feed and topical moxidectin quarantine protocol and found that time or location played a larger role in alterations of the gut microbiota than quarantine treatments on its composition [36]. A notable difference between that study and ours is that we looked at both sexes, not just female mice. In addition, we used the BPH/5 mouse which harbors comorbidities, hypertension and predisposition for obesity in females.

First, phylum change in fecal communities was analyzed after FBZ in both male and female BPH/5. While there were statistical differences in Firmicutes and Bacteroidetes abundance in BPH/5 males after FBZ, this was not observed in females. Moreover, the ratio of Firmicutes to Bacteroidetes was not altered in either BPH/5 males nor females, who display hallmark obesity phenotypes [23,37]. Furthermore, their body weights during the 5-week treatment period (average body weight: male 25.26 ± 0.4180g and female 23.17± 0.4792) were similar to historical age-matched BPH/5 body weights [17,23]. Alterations in their gut microbiome due to treatment may have downstream effects of gut-derived metabolites. The gut microbiome influences short chain fatty acids that effect the overall energy homeostasis and cardiovascular health and disease [38,39]. This is an area that is currently under investigation in the laboratory. Next, Verrucomicrobia and Actinobacteria were significantly altered with FBZ treatment. Verrucomicrobia was increased and was significantly different by sex and treatment interaction. This demonstrates that the treatment had an effect in a sex dependent manner. In a similar study by Gorla et al, Verrucomicrobia increased with the use of an antiparasitic agent [40], which was mirrored in our study. Additionally, Actinobacteria was another phylum that was found to decrease post-treatment using the clr method in this study. In a study by He et al, Actinobacteria decreased with the treatment of FBZ in the Amur tiger [41].

When investigating Verrucomicrobia at the genus level, Akkermansia was found in high abundances. Akkermansia (Verrucomicorbia) was significantly increased in males post treatment and was greater in males compared to females (Fig 2). Denoting the sex specific change at the genus level. In humans, Akkermansia is involved in glucose metabolism and obesity [42]. Enterorhabdus from the phylum Actinobacteria was overrepresented and reduced post treatment in both sexes. Enterorhabdus is related to inflammation and obesity in other mice strains [43,44].

A healthy gut is characterized by high diversity [45]. Alpha diversity which is a measure of evenness and richness was decreased post FBZ in both male and female BPH/5 fecal communities. Thus, demonstrating a potential microbial dysbiosis with FBZ in BPH/5 mice. Beta diversity which is a measure of community structure was altered with FBZ in BPH/5 mice and this emphasizes that treatment potentially causes a microbial community shift.

The BPH/5 females showed a marked decrease in Muribaculaceae spp.,but more clinically relevant is the profound decrease in Lactobacillus found in the BPH/5 female post-treatment. Lactobacillus is a known genusthat promotes healthy intestinal function and reduces inflammation [46–48]. In future microbiome investigations in BPH/5, it will be important to measure Lactobacillus as a potential biomarker of cardiometabolic health and disease. A limitation of this study was not directly measuring the effects of FBZ on blood pressure or obesity in the mice sampled for this study. However, in house colony mice used for blood pressure studies before [18] and post FBZ [17,24], still exhibited high mean arterial pressure. This suggests that treatment did not change the hypertensive phenotype in these mice and their hallmark co-morbidity was maintained.

Although FBZ is not typically considered antibacterial, it has been shown to influence bacterial antimicrobial resistance [49]. Changes in the gut microbiome can affect experimental outcomes(5). In this study, male mice demonstrate decreases of Lachnoclostridium spp, Lachnospiraceae spp, Odoribacter spp, Clostridiales family Xiii ssp, Alistipes spp, and Muribaculceae ssp. The important contributors here are Clostridiales family Xiii ssp, Alistipes spp, and Muribaculceae ssp. Clostridiales family has been previously described to decrease with the use of antibiotics [50] and also decreases with the onset of colitis [51]. Muribaculceae has also been shown to decrease in abundance with the development of colitis [52]. While, Alistipes shows protective attributes in colitis, cancer, and cardiovascular disease [53]. These are main contributors found in this study that may implicate a gut microbiome shifting towards dysbiosis.

In conclusion, this study presents that there are changes in the gut microbial communities after FBZ treatment. These results were different by sex, herein because Akkermansia (Verrucomicrobia) had a greater effect on the males post treatment. Significant changes at the ASV level were also different by sex. Additionally, 16S metagenomic sequencing is relatively insensitive when compared to a complete genomic analysis. Although, the microbial alterations should be a consideration for investigators if FBZ treatment is warranted during experiments.

Abbreviations

FBZ

Fenbendazole

BPH

Blood pressure high

ASV

amplicon sequence variant

PERMANOVA

Permutational multivariate analysis of variance

FDR

False Discovery Rate

clr

centered log ratios

Data Availability

All raw sequence reads, and corresponding metadata can be found on the SRA website accession number PRJNA851094.

Funding Statement

National Institutes of Health (NIH) P20GM135002; Louisiana State University Veterinary Clinical Sciences CORP grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Christopher Staley

31 Jan 2023

PONE-D-23-00107Effects of fenbendazole on fecal microbiome in BPH/5 mice, a model of hypertension and obesity, a brief reportPLOS ONE

Dear Dr. Sones,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Both reviewers found the study to be of value; however, additional clarification of the results is necessary. Furthermore, the discussion and conclusions should be edited to match the data generated in this study, as indicated by the reviewers.

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Academic Editor

PLOS ONE

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Comments to the Author

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Abstract: line 42, define FBZ.

Please clearly mention which comparisons (gender, medication, before/after medication, obesity) are you targeting. Each result line in mentioned for a different comparison. Please give

Give Bray-Curtis dissimilarity p value. The difference is only between male and female which means treatment has no effect. Please give each p values. Results are not coherent with the conclusions.

I wouldn’t say Lactobacillus as a marker of healthy gut. Lactobacillus at genus level has more complex role.

Methods:

What we mean by Adult (8 weeks-6 months of age)?

Line 86 to 91: break in smaller sentences.

Line 102, 103: were not are

Line 107 : exiting the room, respectively

Line 109: Samples collection:

Line 110: individually in sterile empty cages

Line 101: were placed in sterile tubes

Line 129: Microbial community

Changes at the phyla level in BPH/5 mice with Fenbendazole treatment

Herein just mention treatment effects (FBZ). Or if you want to add gender as well, make a crystal clear difference from the start.

Only mention changes with p value < 0.05 or elsewhere mention p value. Result sentences are not written properly, and each sentence has multiple results with no clear reflection. Sometimes past and present are mixed (164-165).

Either figures are not clear, or p values are not mentioned. For figure 2. C, D, and E, use the same headings. As I can see that the post-male has larger bell size, this may lead to non-significant results. Lines 181 to 184: reflect that the effect is due to treatment which contradicts with the conclusions. Overall, I believe that we shall rewrite results with more coherent text and figures.

179: dissimilarity

Discussion:

Line 203: What we mean by institution?

Figure 2 A and B; also use the similar titles. In PCoA I can see that post treatment clustering is poor, which needs to be justified.

Reviewer #2: You should acknowledge as a limitation that although you found some differences in microbial frequency, you did not evaluate effects on blood pressure or obesity, which are key features of the animal model you studied. Also, 16S rRNA testing is relatively insensitive compared to more complete genomic analyses.

**********

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Reviewer #1: Yes: Muhammad Umar Sohail

Reviewer #2: No

**********

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PLoS One. 2023 Jun 9;18(6):e0287145. doi: 10.1371/journal.pone.0287145.r002

Author response to Decision Letter 0

15 Feb 2023

Effects of fenbendazole on fecal microbiome in BPH/5 mice, a model of hypertension and obesity

Beckers et al

Thank you for reviewers’ comments, see red lined below. We have rearranged the discussion per editor’s suggestion to better link results to discussion points.

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Abstract: line 42, define FBZ.

Thank you, we have corrected the mistake and added the definition on line 31.

Please clearly mention which comparisons (gender, medication, before/after medication, obesity) are you targeting. Each result line in mentioned for a different comparison. Please give

A sentence was added to the abstract line 36-38 to aid in clarification. The main purpose of the study was to analyze fecal microbiome communities before and after medication, but it was noted that the alteration were made in a sex dependent manner.

Give Bray-Curtis dissimilarity p value. The difference is only between male and female which means treatment has no effect. Please give each p values. Results are not coherent with the conclusions.

P values have been added to the abstract and results edited, lines 39-44

I wouldn’t say Lactobacillus as a marker of healthy gut. Lactobacillus at genus level has more complex role.

Removed from the abstract

Methods:

What we mean by Adult (8 weeks-6 months of age)?

Thank you, verbiage was added to clarify.

Line 86 to 91: break in smaller sentences.

Corrected, thank you.

Line 102, 103: were not are

Corrected.

Line 107 : exiting the room, respectively

Added, thank you.

Line 109: Samples collection:

Corrected, thank you.

Line 110: individually in sterile empty cages

Corrected, thank you.

Line 101: were placed in sterile tubes

Corrected, thank you.

Line 129: Microbial community

Corrected, thank you.

Changes at the phyla level in BPH/5 mice with Fenbendazole treatment

Herein just mention treatment effects (FBZ). Or if you want to add gender as well, make a crystal clear difference from the start.

Only mention changes with p value < 0.05 or elsewhere mention p value. Result sentences are not written properly, and each sentence has multiple results with no clear reflection. Sometimes past and present are mixed (164-165).

P values have been added to results and result section has been edited to make things clear.

Either figures are not clear, or p values are not mentioned. For figure 2. C, D, and E, use the same headings. As I can see that the post-male has larger bell size, this may lead to non-significant results. Lines 181 to 184: reflect that the effect is due to treatment which contradicts with the conclusions. Overall, I believe that we shall rewrite results with more coherent text and figures.

Thank you for pointing out this flaw. Titles are corrected on all figures to the same headings and p-values have been added to each figure as well.

179: dissimilarity

Corrected, thank you.

Discussion:

Line 203: What we mean by institution?

The intended meaning was location of where those mice were housed. Corrected in text, thank you.

Figure 2 A and B; also use the similar titles. In PCoA I can see that post treatment clustering is poor, which needs to be justified.

Titles have been edited to be similar. Clustering is poor due to the hypothesis that the treatment affected the males more greatly cause a greater community change in the post treatment samples.

Reviewer #2: You should acknowledge as a limitation that although you found some differences in microbial frequency, you did not evaluate effects on blood pressure or obesity, which are key features of the animal model you studied. Also, 16S rRNA testing is relatively insensitive compared to more complete genomic analyses.

Thank you for the input. This is a good point and has been added to the discussion on lines 240-243.

Attachment

Submitted filename: Comments_plosOne_FBZ_2023.docx

Click here for additional data file. (16KB, docx)

Decision Letter 1

Christopher Staley

3 Apr 2023

PONE-D-23-00107R1Effects of fenbendazole on fecal microbiome in BPH/5 mice, a model of hypertension and obesity, a brief reportPLOS ONE

Dear Dr. Sones,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by May 18 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Christopher Staley, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments :

Following review by two new reviewers, additional minor clarifications are requested. Importantly, sex differences and confounding factors should be accounted for where possible in the analyses, as suggested by the reviewer. Where this is not possible, it should be explained and explored in the text.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: All comments have been addressed

Reviewer #4: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Yes

Reviewer #4: No

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

Reviewer #4: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: Yes

Reviewer #4: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: The manuscript by Beckers et al. addresses the effect of FBZ anti-parasitic treatment on stool microbiota in a mouse model of hypertension and obesity. This manuscript is well written and the analyses are straightforward. I reviewed the R1 version of the manuscript which was a response to the comments from 2 other reviewers. My comments do not take into account these earlier comments and I leave it to the editor which of my comments should be addressed. I do have a few comments:

One interesting aspect was not addressed in the discussion. These mice are inbred and are housed and fed in a very controlled environment. Therefore, I would expect (although I am not very experienced in the laboratory mouse stool microbiome) that inter-individual differences are not as high as presented in Fig. 1. If the number of words allows, I would like to see some comments on that in the discussion.

Another interesting aspect that was not clearly addressed. This is longitudinal data. The analyses can make use of these differences before and after treatment for each animal. That can be done for alphas as well as single genera. Was this done in the two way ANOVA analyses? Please comment on this.

Third interesting aspect that was not addressed. Phylum Verrucomicrobiota was higher in males after treatment which was not observed in females. The differential abundance analyses at genus-level did not show what genera were responsible for this. Please discuss why. Further, in humans Verrucomicrobiota (especially genus Akkermansia) are known to be involved in glucose metabolism and obesity. Also, genus Akkermansia is higher in females. This might be interesting in combination with the phenotype of these mice. If the number of words allows, I would like to see some comments on that in the discussion.

Analyses have been performed either at phylum-level or at genus-level, if I am correct. It is not clear how taxa were aggregated to these higher levels and it is not clear at what levels the analyses were performed, i.e., Bray-Curtis dissimilarity and differential abundances. Also, in lines 188 and 189, ASV-level analyses were mentioned but ASVs are basically at strain-level. Please make clear.

Line 115, please indicate if bead beating was part of the protocol.

Line 125, the protocol in the reference is not the manufacturer’s guideline. Illumina has another protocol. Please make sure the correct reference is given.

In line 140 it is stated that normality was confirmed, but most ASVs are not normally distributed. Please explain.

In line 141, I assume that “both p values and FDR-corrected p values <0.05 were considered significant”.

Throughout the text, taxonomic levels are written in plural form which is not always correct, e.g., phyla instead of phylum (lines 146, 149) and genera instead of genus (line 234).

Sentence in line 138 is unclear.

Reviewer #4: In this article, the authors investigated the effects of Fenbendazole on the gut microbiome of mice with hypertension and reported the difference in gut microbiome responses to the treatment between male and female mice.

This article reported an observation without controls over baseline status or any confounding factors, such as BMI, in female and male mice. The main conclusion “there are changes in the gut microbial communities after FBZ use in a sex dependent manner” is not very convincing with current study design or evidence present in the article.

Major issues:

1. Difference between baseline microbiome in male and female mice

According to figure2, there are sex-related differences at baseline. Microbiome components interact between each other. The observation of change in one single component might have multiple components involved. Plus the regression toward the mean effects in statistics. If the baseline microbiome was different between sex, then we will expect the change will be different. The authors need to calibrate for the baseline difference.

2. Lack of controls over confounding factors

As mentioned by the authors, obesity has a great influence on gut microbiome and the treatment may have sex-dependent effects on obesity. Is there a difference in BMI between female and male mice at baseline? Is there a difference in the change of BMI between female and male mice during treatment? These were not reported.

3. Statistical analysis

This study collected fecal samples at two time points from each mice. Using baseline microbiome as reference and comparing within subject changes will be a better choice than current methods, for example difference in difference model. The authors may also consider propensity score matching to remove effects of confounding factors.

4. More details in “Changes at the ASV level with Fenbendazole treatment and phenotypic outcomes”

“Changes at the ASV level with Fenbendazole treatment and phenotypic outcomes” worth more emphasis than “Changes at the phyla level in BPH/5 mice with Fenbendazole treatment”. ASV level is more informative than phyla level in showing microbiome functions.

Minor issues:

1. “Treatment had a significant effect on community composition using PERMANOVA of the Bray-Curtis dissimilarity matrix, with both BPH/5 female and male microbial communities differing post-treatment but not different by sex or interaction (treatment p=0.002, sex p=0.3, interaction p=0.05) (Figure 3b).” For “ sex p=0.3”, what was compared? Female and male samples with both time points pooled together? This analysis is actually showing that treatment had much bigger effects than sex, not supporting the conclusion.

2. The resolution of figures is too low.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: No

Reviewer #4: No

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PLoS One. 2023 Jun 9;18(6):e0287145. doi: 10.1371/journal.pone.0287145.r004

Author response to Decision Letter 1

8 May 2023

Effects of fenbendazole on fecal microbiome in BPH/5 mice, a model of hypertension and obesity

Beckers et al

Thank you for reviewers’ comments, see red lined below. We have rearranged the discussion per editor’s suggestion to better link results to discussion points.

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: The manuscript by Beckers et al. addresses the effect of FBZ anti-parasitic treatment on stool microbiota in a mouse model of hypertension and obesity. This manuscript is well written and the analyses are straightforward. I reviewed the R1 version of the manuscript which was a response to the comments from 2 other reviewers. My comments do not take into account these earlier comments and I leave it to the editor which of my comments should be addressed. I do have a few comments:

One interesting aspect was not addressed in the discussion. These mice are inbred and are housed and fed in a very controlled environment. Therefore, I would expect (although I am not very experienced in the laboratory mouse stool microbiome) that inter-individual differences are not as high as presented in Fig. 1. If the number of words allows, I would like to see some comments on that in the discussion.

Even though these mice are inbred and were housed in the same environment, some individual variation is noted on the bar graph of Figure 1, but nothing was significantly different when the individuals were used as the random effect in the 2-way ANOVA. The only thing to note of significance was the phyla Actinobacteria and Verrucomicrobia when assessing the time points pre and post, which is presented in Figure 2.

Another interesting aspect that was not clearly addressed. This is longitudinal data. The analyses can make use of these differences before and after treatment for each animal. That can be done for alphas as well as single genera. Was this done in the two way ANOVA analyses? Please comment on this.

A 2-way ANOVA was performed with time, sex, time x sex as the fixed effects and each mouse as the random effect. This was followed up with Post hoc and Fisher’s LSD for additional analysis. A clarification was added to the statistics section within the methods to further explain. Thank you. Please see line 139-142.

Third interesting aspect that was not addressed. Phylum Verrucomicrobiota was higher in males after treatment which was not observed in females. The differential abundance analyses at genus-level did not show what genera were responsible for this. Please discuss why. Further, in humans Verrucomicrobiota (especially genus Akkermansia) are known to be involved in glucose metabolism and obesity. Also, genus Akkermansia is higher in females. This might be interesting in combination with the phenotype of these mice. If the number of words allows, I would like to see some comments on that in the discussion.

Akkermansia was the genus of Verrucomicrobiota found in this study. This information was added to the discussion lines 232-235. Thank you.

Analyses have been performed either at phylum-level or at genus-level, if I am correct. It is not clear how taxa were aggregated to these higher levels and it is not clear at what levels the analyses were performed, i.e., Bray-Curtis dissimilarity and differential abundances. Also, in lines 188 and 189, ASV-level analyses were mentioned but ASVs are basically at strain-level. Please make clear.

Thank you for identifying this confusion, it was clarified within the methods section.

Line 115, please indicate if bead beating was part of the protocol.

A clarification has been added on line 118, thank you.

Line 125, the protocol in the reference is not the manufacturer’s guideline. Illumina has another protocol. Please make sure the correct reference is given.

An additional reference has been added.

In line 140 it is stated that normality was confirmed, but most ASVs are not normally distributed. Please explain.

This statement was removed, it was in regard to additional data that was previously removed upon last reviewers request.

In line 141, I assume that “both p values and FDR-corrected p values <0.05 were considered significant”.

The statement within the methods was revised.

Throughout the text, taxonomic levels are written in plural form which is not always correct, e.g., phyla instead of phylum (lines 146, 149) and genera instead of genus (line 234).

These grammatical errors were corrected.

Sentence in line 138 is unclear.

This sentence was edited to make it clearer.

Reviewer #4: In this article, the authors investigated the effects of Fenbendazole on the gut microbiome of mice with hypertension and reported the difference in gut microbiome responses to the treatment between male and female mice.

This article reported an observation without controls over baseline status or any confounding factors, such as BMI, in female and male mice. The main conclusion “there are changes in the gut microbial communities after FBZ use in a sex dependent manner” is not very convincing with current study design or evidence present in the article.

Major issues:

1. Difference between baseline microbiome in male and female mice

According to figure2, there are sex-related differences at baseline. Microbiome components interact between each other. The observation of change in one single component might have multiple components involved. Plus the regression toward the mean effects in statistics. If the baseline microbiome was different between sex, then we will expect the change will be different. The authors need to calibrate for the baseline difference.

A clarification and a previous references were added to note that there were no difference between male and females prior to treatment. The main difference observed was in Verrucomicrobia post treatment specifically Akkermansia, which is clarified in the discussion.

2. Lack of controls over confounding factors

As mentioned by the authors, obesity has a great influence on gut microbiome and the treatment may have sex-dependent effects on obesity. Is there a difference in BMI between female and male mice at baseline? Is there a difference in the change of BMI between female and male mice during treatment? These were not reported.

One of the reviewers on the initial submission requested that we remove this information. Therefore, we will keep it omitted at this time.

3. Statistical analysis

This study collected fecal samples at two time points from each mice. Using baseline microbiome as reference and comparing within subject changes will be a better choice than current methods, for example difference in difference model. The authors may also consider propensity score matching to remove effects of confounding factors.

Based on comments from reviewer 3, we used a mixed ANOVA with repeated measures for the analysis, no difference was found between sex pre-treatment.

4. More details in “Changes at the ASV level with Fenbendazole treatment and phenotypic outcomes”

“Changes at the ASV level with Fenbendazole treatment and phenotypic outcomes” worth more emphasis than “Changes at the phyla level in BPH/5 mice with Fenbendazole treatment”. ASV level is more informative than phyla level in showing microbiome functions.

More detail was added to this section of the results, specifically the genera involved in the changes in Actinobacteria and Verrucomicrobia.

Minor issues:

1. “Treatment had a significant effect on community composition using PERMANOVA of the Bray-Curtis dissimilarity matrix, with both BPH/5 female and male microbial communities differing post-treatment but not different by sex or interaction (treatment p=0.002, sex p=0.3, interaction p=0.05) (Figure 3b).” For “ sex p=0.3”, what was compared? Female and male samples with both time points pooled together? This analysis is actually showing that treatment had much bigger effects than sex, not supporting the conclusion.

This was clarified in the last paragraph of the discussion, lines 271-273.

2. The resolution of figures is too low.

Attachment

Submitted filename: Commets_v2_plosone_FBZ_2023 (1).docx

Click here for additional data file. (18.3KB, docx)

Decision Letter 2

Christopher Staley

31 May 2023

Effects of fenbendazole on fecal microbiome in BPH/5 mice, a model of hypertension and obesity, a brief report

PONE-D-23-00107R2

Dear Dr. Sones,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Christopher Staley, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

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Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #3: Yes

Reviewer #4: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #3: Yes

Reviewer #4: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: My comments have been addressed properly, therefore, I recommend to accept this version of the manuscript.

Reviewer #4: The authors have explained thoroughly for the confusions and all the comments have been addressed. Thank you!

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Reviewer #3: No

Reviewer #4: No

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Acceptance letter

Christopher Staley

2 Jun 2023

PONE-D-23-00107R2

Effects of fenbendazole on fecal microbiome in BPH/5 mice, a model of hypertension and obesity, a brief report

Dear Dr. Sones:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

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on behalf of

Dr. Christopher Staley

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    Attachment

    Submitted filename: Comments_plosOne_FBZ_2023.docx

    Click here for additional data file. (16KB, docx)
    Attachment

    Submitted filename: Commets_v2_plosone_FBZ_2023 (1).docx

    Click here for additional data file. (18.3KB, docx)

    Data Availability Statement

    All raw sequence reads, and corresponding metadata can be found on the SRA website accession number PRJNA851094.

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