Influence of Debaryomyces hansenii on bacterial lactase gene diversity in intestinal mucosa of mice with antibiotic-associated diarrhea

Yunshan He; Yuan Tang; Maijiao Peng; Guozhen Xie; Wenge Li; Zhoujin Tan

doi:10.1371/journal.pone.0225802

. 2019 Dec 6;14(12):e0225802. doi: 10.1371/journal.pone.0225802

Influence of Debaryomyces hansenii on bacterial lactase gene diversity in intestinal mucosa of mice with antibiotic-associated diarrhea

Yunshan He ¹, Yuan Tang ¹, Maijiao Peng ¹, Guozhen Xie ¹, Wenge Li ^2,^*, Zhoujin Tan ^1,^*

Editor: Zongxin Ling³

PMCID: PMC6897403 PMID: 31809511

Abstract

Aim

The current study aimed to investigate the effects of Debaryomyces hansenii on the diversity of bacterial lactase gene in the intestinal mucosa of antibiotic-associated diarrhea (AAD) mice.

Methods

Eighteen mice were randomly divided into three groups (6 mice per group): healthy control group, diarrhea model group and D. hansenii treatment group. The antibiotic-associated diarrhea model was established by intragastric administration with a mixture of cephradine and gentamicin sulfate (23.33 mL·kg^-1·d^-1) twice a day for 5 days continuously. After establishing the AAD model, the mice in the D. hansenii treatment group were gavaged with D. hansenii for three days, while other groups were gavaged with distilled water. Then, the intestinal mucosa of all three groups was collected and DNA was extracted in an aseptic environment for the following analysis.

Results

The difference in the richness and homogeneity of the bacterial lactase gene among all samples were inapparent, as the difference in the Chao1, ACE, Simpson and Shannon indices among the three groups were insignificant (P>0.05). NMDS analysis also showed that the distance of the samples among the three groups was unobvious. Furthermore, the bacterial lactase gene in the mucosa mainly originated from Actinobacteria, Firmicutes and Proteobacteria. Compared with the healthy control group, the abundance of lactase genes originating from Cupriavidus, Lysobacter, Citrobacter, Enterobacter and Pseudomonas was increased in the D. hansenii treatment group, while the lactase gene from Acidovorax and Stenotrophomonas decreased (p < 0.01 or p < 0.05) in the diarrhea model group and the D. hansenii treatment group.

Conclusion

D. hansenii was capable of improving the growth of some key lactase-producing bacteria like Deinococcus, Cupriavidus and Lysobacter for treating AAD.

Introduction

Lactase, also known as β-galactosidase, is an important enzyme for intestinal function, which can hydrolyze lactose into glucose and galactose, allowing lactose to be absorbed and utilized by human beings and animals. Because of inactivity and deficiency of lactase, lactose is fermented by intestinal bacteria to produce significant amounts of short-chain fatty acid (SCFA) and hydrogen rather than hydrolyzed, which cause diarrhea and is referred to as lactose intolerance (LI) [1,2]. Usually, intestinal lactase is produced by intestinal epithelial cells, microorganisms and obtained from exogenous lactase[3]. Nevertheless, with the widespread use of antibiotics in the clinic, increasing attention has been paid to antibiotic-associated diarrhea (AAD). Currently, it is believed that AAD, persistent diarrhea and lactase-deficiency diarrhea are all related to low lactase activity, which is probably due to a reduced number of lactases, affecting the arrangement and shedding of villi[4]. Fortunately, most diarrhea can be relieved by oral probiotics such as Lactobacillus, Bifidobacterium, Saccharomyces boulardii and by lactase supplementation [5–8].

Approximately 1000 species of bacteria inhabit the human intestinal tract, which plays an important role in metabolism, immunity and other physiological functions[9,10]. The intestinal microbes that inhabit the enteric cavity and epithelial cells of the mucosa establish a complete intestinal mucosal barrier together with intestinal epithelial cells, mucus and secrete so as to play a critical role in maintaining the homeostasis and healthy[11,12]. However, the extensive use of antibiotics pose a risk of improper application, which could cause an imbalance in the intestinal microbiota, destruction of the mucosal barrier related diseases and eventually[13]. Our previous research reported that antibiotics decrease the diversity of bacterial lactase genes in the intestinal contents, and they transforme their community structures[14].

Yeast has been widely used in medicine, food, beverage, alcohol and other industries because of its rapid propagation ability and efficient metabolism[15]. D. hansenii, isolated from the natural environment, food or intestinal tract, is one of the most important unconventional yeasts. A large number of studies verified that D. hansenii plays an important role in the fermentation of cheese and sausage and the production of fuel alcohol. It can produce critical esters in sausage manufacture, as well as the thermophilic lactase necessary to produce fuel alcohol[16–20]. In addition, it can also produce antitoxins to inhibit the growth of the harmful bacteria such as Candida [21]. Our previous studies showed that D. hansenii, which was isolated from the mouse intestine[19], was able to tolerate a high acid and high bile salt environment, and it had high viability in the artificial gastrointestinal fluid environment. In addition, the combination of 25% D. hansenii and 25% Qiweibaizhusan was used to modulate the population of total intestinal bacteria and Escherichia coli, and it can also restore the bacterial diversity in mice with dysfunctional diarrhea[22–25]. The vast majority of previous studies focused on the pathogenesis or treatment of AAD, and our previous studies reported the influence of D. hansenii on AAD based on intestinal bacterial diversity as well. The current research aimed to investigate the effect of D. hansenii on AAD based on the diversity of the bacterial lactase gene in intestinal mucosa by using high-throughput sequencing. It can provide further experimental basis for the development and utilization of D. hansenii as a new microecological preparation.

Materials and methods

Reagents and medicine

Cephradine capsules (batch number: 151101) were purchased from Suzhou Chung-Hwa Chemical & Pharmaceutical Industrial Co. Ltd., and gentamicin sulfate (batch number: 5150307) was purchased from Yichang Pharmaceutical Industrial Co. Ltd. The two antibiotics were then prepared into a mixture at a concentration of 62.5 g·L^-1[26]. The DNA extraction reagents (such as Proteinase K, lysozyme, Tris-saturated phenol-chloroform-isoamyl alcohol (25:24:1) and TE buffer) were purchased from Beijing Dingguo Changsheng Biotechnology Co. Ltd., and prepared in the lab (such as 10% SDS, chloroform-isoamyl alcohol (24:1), 5 mol·L^-1 NaCl, 0.1 mol·L^-1 PBS and CTAB/NaCl). D. hansenii, which was provided by the laboratory and shaken at 28°C for 36 hours after being inoculated into liquid Potato Sucrose medium in a 300 mL erlenmeyer flask. The cells were then gathered by centrifugation at 2000×g for 4 minutes after washed 1∼2 times repeatedly with sterile stroke-physiological saline solution. The above cells were diluted to 10¹⁰ mL^-1 with sterile storke-physiological saline solution eventually after being counted by hemocytometer, and stored at 4°C for subsequent experiments[27].

Animals and procedures

Eighteen specific pathogen-free (SPF) Kunming (KM) mice (nine male and nine female, one month old), weighting 20±2 g, were purchased from Hunan Slaccas Jingda Laboratory Animal Company with license number SCXK (Xiang) 2013–0004. All procedures involving animals were performed according to protocols approved by the Institutional Animal Care and Use Committee of Hunan University of Chinese Medicine. Mice were randomly divided into three groups (6 mice per group, half male and half female): healthy control group (tlcm), diarrhea model group (tlmm) and D. hansenii treatment group (tljm). Mice in the healthy control group were gavaged with 0.35 mL of sterile water twice a day for 5 days. To induce diarrhea, mice in both the diarrhea model group and the D. hansenii treatment group were injected intragastrically with a mixture of gentamicin sulfate and cefradine capsules (23.33 mL·kg^-1·d^-1) twice a day for 5 days by following the procedures described previously[28]. After diarrhea symptoms appeared (such as erected coat, reduced intake, watery stool and declined activity), the mice in the D. hansenii treatment group were treated with D. hansenii by intragastric administration (0.35 mL, 10¹⁰ mL^-1), and the other two groups were given aseptic water twice a day for 3 days. Then, the mucosa from the jejunum to ileum were scraped with a cover slip after the contents were extruded and the intestine was washed twice with sterile saline solution in a germ-free environment (each sample contains mucosa from two mice: one male and the other female, which has three samples per group), and then were frozen immediately and stored at -20°C for further use [29].

Ethical approval was obtained from the Animal Ethics and Welfare Committee of Hunan University of Chinese Medicine.

Total DNA extraction from intestinal mucosa

Total DNA from the intestinal mucosa was extracted following to our previous reports[30,31]. A total of 2.0 g of mucosa was weighed in a germ-free environment and preprocessed with 0.1 mol·L^-1 phosphate buffer solution (PBS) and acetone. The cells were collected by the above method and resuspended in 4 mL of TE buffer. Cells walls were disrupted by lysozyme, and total DNA was purified and extracted by proteinase K, CTAB/NaCl, Tris saturated phenol-chloroform-isoamyl alcohol (25:24:1), chloroform-isoamyl alcohol (24:1), absolute ethyl alcohol and sodium acetate after cells wall-broken by lysozyme. Eventually the DNA was dissolved in 50 μL of TE buffer for further analysis.

PCR amplification of the mucosal bacterial lactase gene and high-throughput sequencing

The universal primers for PCR amplification were designed according to the lactase gene sequences of Lactobacillus and Escherichia coli from NCBI and synthesized by Shanghai Personal Biotechnology Co., Ltd.[32]. The forward primer was 5'-TRRGCAACGAATACGGSTG-3', and the reserve primer was 5'-ACCATGAARTTSGTGGTSARCGG-3'. After PCR mixtures were prepared (25 μL) (including 5 μL of 5 × high GC buffer, 0.25 μL of Q5 high-fidelity DNA polymerase, 1 μL of 10 μmol·L^-1 forward primer, 0.5 μL of 10 mmol·L^-1 dNTP, 1 μL of 10 μmol·L^-1 reserve primer, 1 μL of DNA template and 11.25 μL of sterilized ddH₂O) and added to 0.5 mL PCR tubes, the amplification was carried out as follows: conditions: initial denaturation at 98°C for 30 s, 32 cycles of denaturation at 98°C for 15 s, annealing at 46°C for 30 s and extension at 72°C for 30 s, then extension at 72°C for 5 min and holding at 4°C. PCR products of the bacterial lactase gene were purified and then detected using high-throughput sequencing, which was performed by Shanghai Personal Biotechnology Co., Ltd.

Gene diversity and statistical analysis

The software available online, including QIIME (http://qiime.org/)[33] and Mothur (http://www.mothur.org/), were used to analyze the sequencing results. Alpha diversity analysis, including Chao1, ACE, Simpson and Shannon indices, was applied to identify the richness and uniformity of the intestinal mucosa bacterial lactase gene by determining the operational taxonomic units (OTUs)[34–37]. Principle component analysis (PCA)[36] was used to analyze the community difference of lactase-producing bacteria according to the distance among individuals. The source and abundance of the bacterial lactase gene at the specific taxonomic levels are presented in the figure containing species evolution and abundance information and statistics table of relative abundance statistics table. Then, our measurement data were analyzed using the SPSS 21.0 software (IBM Corp, Armonk, NY, USA), of which one-way ANOVA was applied to compare the statistical significance of differences, with p value.

Results

Sequence statistics and OTU analysis

The diversity and richness of lactase gene can be well studied by measuring and analyzing OTUs (operational taxonomic unit). Fig 1 shows that the numbers of OTUs of the healthy control group, diarrhea model group and D. hansenii treatment group was 298, 435 and 326, respectively. The results showed that 45 OTUs were unique to the healthy control group, 202 OTUs were unique to the diarrhea model group and 57 OTUs were unique to the D. hansenii treatment group. These results suggested that antibiotic modeling increased the number of OTUs of the lactase gene from the intestinal mucosal bacteria in mice, while the number of OTUs of antibiotic models can be returned to normal following treatment with D. hansenii.

Alpha diversity of the bacterial lactase gene from the intestinal mucosa of AAD mice treated with D. hansenii

By drawing rarefaction curves, the diversity of each sample could be measured to some extent. Fig 2 shows that each curve tended to flatten with the increase in the number of measured sequences. This result suggested that the sequencing results were more than enough to reflect the current sample containing the intestinal mucosa lactase gene diversity.

The rank abundance curve and alpha indices can be used to determine the richness and uniformity of the bacterial lactase gene from intestinal mucosa to evaluate the therapeutic efficacy of D. hansenii treatment on AAD. As shown in Fig 3, there was no significant difference in the length on abscissa and the gentleness, which indicated that D. hansenii treatment had no significant impact on the richness and uniformity of the bacterial lactase gene among the healthy control group and diarrhea model group. From the alpha indices, we determined that there was no significant difference in the Chao1, ACE, Simpson and Shannon indices among the three groups (Table 1).

Fig 3 — Note: The abscissa represents the ordinal of the OTU, and the ordinate represents the abundance of the OTU. The larger the curve span, the richer the composition of the species was. The flatter the curve, the higher the evenness the species composition was. tlcm 1–3 represented the healthy control group samples 1–3, tlmm1-3 represented the diarrhea model group samples 1–3, tljm 1–3 represented the D. *hansenii* treatment samples 1–3.

Table 1. Alpha diversity indices of the bacterial lactase gene from the intestinal mucosa of antibiotic-associated diarrhea mice treated with D. hansenii.

Group	Chao1	ACE	Shannon	Simpson
tlcm	121.89±8.81	127.93±13.58	0.80±0.18	0.18±0.04
tlmm	160.33±24.15	167.13±13.60	2.26±1.43	0.54±0.29
tljm	150.85±61.02	154.10±58.53	1.45±0.38	0.38±0.05

Open in a new tab

Note: The larger Chao1 and ACE indices represent higher richness of the bacterial lactase gene. The larger Shannon and Simpson index a represents higher diversity of bacterial lactase genes. tlcm, tlmm and tljm represented the healthy control group, diarrhea model group, and D. hansenii treatment group, respectively.

Beta diversity of bacterial lactase genes in the intestinal mucosa of AAD diarrhea mice treated with D. hansenii

PCA(principal component analysis) analysis can extract the most important differences between samples from the original data. As shown in Fig 4, that the three samples in the healthy control group and the D. hansenii treatment group were relatively close to each other, while the distance between the groups was relatively far, which can be clearly distinguished. The distribution of the three samples in the diarrhea model group was very scattered and not completely separated from the healthy control group and the D. hansenii treatment group. This finding suggests that antibiotics altered the structure of bacterial lactase-producing genes in the intestinal mucosa, and the percentages attributed to the variations in PC1 and PC2 were 97.32% and 2.66%, respectively.

The differences in the community were measured by distance comparison among individuals through NMDS analysis. The distribution of samples in the healthy control group was more concentrated than that in the other groups, and the distance between the diarrhea model group and the healthy control group was approximately the same distance as that between the D. hansenii treatment group and the healthy control group (Fig 5). The results indicated that D. hansenii treatment had no significant effect on the recovery of the community structure of the bacterial lactase gene from the intestinal mucosa.

Fig 5 — Different color points represent samples from different groups. The closer distance between the two points represented the smaller the community difference of the lactase-produced bacteria between the two samples. tlcm, tlmm and tljm represent the healthy control group, the diarrhea model group and the D. *hansenii* treatment group respectively.

Abundance and source of the bacterial lactase gene from the intestinal mucosa of AAD mice treated with D. hansenii

As shown in Fig 6(A), at the genus level, the number of known lactase-producing bacteria detected in the healthy control group, diarrhea model group and D. hansenii treatment group were 16, 12, and 16, respectively. Mesorhizobium was only found in the healthy control group, and Plasmodium was only detected in the D. hansenii treatment group, Sphingomonas and Bordetella were not detected in the diarrhea model group. In addition, a number of other bacterial lactase genes (other, including some genera with low abundance or unclassified) and some new lactase-producing bacterial genera (no Blast hits) were detected. Compared with the healthy control group, the abundance of Cupriavidus was increased significantly after treatment with D. hansenii. The lactase genes originating from Acidovorax and Stenotrophomonas were lower in the diarrhea model group and D. hansenii treatment group than the healthy control group. Conversely, the lactase gene originated from Citrobacter, Enterobacter and Pseudomonas in the diarrhea model group and D. hansenii treatment group was lower than that in the healthy control group. However, the lactase genes of five genera showed no significant difference between the diarrhea model group and the D. hansenii treatment group. Through further analysis with LEfSe, shownin Fig 6(B), it was found that Sphingomonadaceae, Sphingomonas and Sphingomonadales were the key colony members in the D. hansenii treatment group, while no key colony members were found in the healthy control group or diarrhea model group.

The species evolution and abundance information were used for source and abundance analysis of the bacterial lactase gene at different classification levels (Fig 7). The evolution tree showed that the bacterial lactase genes in intestinal mucosa mainly originated from Actinobacteria, Firmicutes and Proteobacteria. The abundance of the lactase gene in Actinobacteria from the D. hansenii treatment group was the highest, followed by the healthy control group and the diarrhea model group. Lactase genes from Aeromonas, Curvibacter and Deinococcus were only detected in the D. hansenii treatment group, while those from Enterobacteriaceae and Clostridium were only detected in the diarrhea model group. Compared with the other two groups, the abundance of lactase genes originating from Enterococcus, Lysobacter and Salmonella was the highest in the D. hansenii treatment group.

Fig 7 — The pie chart for each branch node indicated the taxon abundance in each sample;the larger the fan area, the higher the abundance of the bacterial lactase gene. tlcm, tlmm and tljm represented the healthy control group, diarrhea model group and D. *hansenii* treatment group, respectively.

Discussion

The gastrointestinal mucosa is the largest interface where interactions mainly occur between symbiotic bacteria and the host. A dynamic and diverse bacterial community inhabits the mucosa, which is an important part of the intestinal mucosa barrier[38]. As an important metabolic organ, intestinal flora participate in metabolism of human and animal metabolism with various microbial enzymes under gene regulation [39]. Metabolism will be affected as soon as the enzyme activity is absent or reduced. Furthermore, except for dysbacteriosis, the mechanisms of AAD include intestinal epithelial cilia atrophy, intestinal mucosal damage and a decrease in cellular enzyme activity as well [40]. Fortunately, AAD can be prevented through the rational use of medicines, and microecologics has become one of the common methods for treating AAD. Our previous research has proven that 25% ultramicro Qiweibaizhusan combined with 25% yeast has a relatively better therapeutic effect on AAD [24], which proves that yeast has a certain therapeutic effect on AAD. Based on the chemical nature of proteins, the activities of most enzymes are regulated by coding genes and other physicochemical factors. At the same time, with the development of modern biological technology and continuous research on intestinal microbiology, exploring the interactions between intestinal flora and intestinal diseases from the perspective of bacterial functional enzyme genes has become a new approach to conduct intestinal microbial research. Therefore, the current study was conducted to investigate the effect of D. hansenii on the bacterial lactase gene diversity from the intestinal mucosa of AAD mice. The results can be analyzed from three aspects: the variety of lactase-producing bacteria, the abundance of lactase-producing bacteria and the effect of D. hansenii on the mucosal barrier.

Intestinal bacteria mainly consist of Proteobacteria, Mycobacteria, Actinomycetes, Bacteroidetes, Mycobacteria, Cyanobacteria and Fusobacteria, especially Bacteroidetes and Firmicutes, which account for approximately 90% of the total[41]. Our results showed that the lactase gene in intestinal mucosa originated from Actinobacteria, Firmicutes and Proteobacteria, among which Proteobacteria accounted for more than 90%. There are differences in lactase activities produced by different bacteria in the intestine; therefore, bacterial lactase gene diversity could be reflected through differences in the population and variety of lactase-producing bacteria. NMDS analysis showed that antibiotics caused a large difference in the community structure of lactase-producing bacteria, whereas the recovery effect of D. hansenii treatment on the community structure was not obvious. The statistical analysis results of lactase-producing bacteria at different classification levels indicated that the D. hansenii had no significant effect on the population of lactase-producing bacteria in the intestinal mucosa at all classification levels except for the order level.

From the high-throughput sequencing results, the relative abundance was used to reflect the quantity difference in lactase-producing bacteria. Of the known genera, the abundance of the lactase gene originating from Cupriavidus in the healthy control group was significantly higher than that in the D. hansenii treatment group. The study of Ueatrongchit showed that Cupriavidus was the genus that produced various microbial enzymes and determined that Cupriavidus necator produced L-threonine 3-dehydrogenase that catabolizes L-threonine[42]. Lysobacter, which is a genus of bacteria with fast growth, strong resistance and no pathogenicity[43], not only can secrete a variety of extracellular enzymes such as chitinase and β-1,3-glucanase, but also have significant antagonism against various pathogenic bacteria [44]. Our results showed that the lactase gene in Deinococcus was detected only in the D. hansenii treatment group and that the lactase gene in Lysobacter was highly abundant in the D. hansenii treatment group, which suggested that D. hansenii increased the quantity of potentially valuable lactase-producing bacteria in order to treat AAD.

The extended-spectrum cephalosporin resistant (ESC^R) Enterobacteriaceae pose a serious infection control challenge for public health [45]. The appearance of the ESC^R phenotype is mainly promoted through plasmid-mediated lateral extended-spectrum β-lactamases (ESBLs) and AmpC gene transfer within Enterobacteriaceae [46]. The current results showed that the lactase gene in Enterobacteriaceae was detected only in the diarrhea model group. The Lactase gene in Enterobacteriaceae in the diarrhea model group was higher than that in the other groups. Moreover, the abundance of the lactase gene in Enterococcus was the highest in the D. hansenii treatment group, followed by the healthy control group and the diarrhea model group. Enterococcus probiotics can increase the expression of small intestinal mucosa tight junction proteins and activity of toll-like receptors 2, 4 and 9 in small intestinal mucosa, inducing an immune response in piglets [47]. All of these studies suggested that antibiotics damaged the mucosa barrier by increasing the population of bacteria with antibiotic resistance and causing dysbacteriosis. And the mucosal barrier could be restored by D. hansenii.

After treatment with D. hansenii, only a few bacterial lactase genes was recovered or increased. The recovery of lactase gene diversity in the intestinal mucosa of AAD mice was not significant. Perhaps our current results can be explained by a recent report, which determined that antibiotics had a long-term impact on intestinal microorganisms[36]. The lactase-producing bacteria in the intestinal mucosa of the diarrhea model could not return to normal levels after a few days, even following treatment with D. hansenii. Moreover, the activity of digestive enzymes present in the brush border of villous epithelial cells in the small intestine mucosa has the function of repairing intestinal mucosa, which is closely related to the structural integrity of the mucosa and probiotics [48,49]. In conclusion, our results suggested that antibiotics disrupted the intestinal mucosa flora in mice, and treatment with D. hansenii may be effective to treat diarrhea by promoting the growth of a few key lactase-producing bacteria or some beneficial bacteria to repair the intestinal mucosa structure.

Supporting information

S1 Dataset. The raw data of healthy control group (tlcm).

(ZIP)

Click here for additional data file.^{(40.2MB, zip)}

S2 Dataset. The raw data of diarrhea model group (tlmm).

(ZIP)

Click here for additional data file.^{(50.2MB, zip)}

S3 Dataset. The raw data of D. hansenii treatment group (tljm).

(ZIP)

Click here for additional data file.^{(24.8MB, zip)}

Data Availability

All relevant data are within the manuscript.

Funding Statement

The research was supported by the National Natural Science Foundation of China (No.81573951) to ZT. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0225802.r001

Decision Letter 0

Zongxin Ling

23 Aug 2019

PONE-D-19-19505

Influence of Debaryomyces hansenii on bacteria lactase gene diversity in intestinal mucosa of mice with antibiotic-associated diarrhea

PLOS ONE

Dear Professor Tan,

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.

According to the comments from the two reviewers, the manuscript has serious flaws including writing, statistical analysis, and data demonstration.

1. You should revise your language with a native English speaker. 2. You should upload your raw data into public database such as NCBI and provide the accession number. Table 1 should be deleted as the data could be gained according to your raw data. 3. For data analysis, you should perform data normalization, i.e. each sample with same read numbers, before the subsequent data analysis.

4. Alpha diversity analysis should included richness indices and diversity indices. Please added these indices.

5. For PCoA analysis, you should include different algorithms such as weighted UniFrac, unweighted UniFrac and Bray-Curtis.

6. For differential bacteria analysis, you should perform LEfSe analysis.

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Zongxin Ling

Academic Editor

PLOS ONE

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Comments to the Author

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Reviewer #1: Partly

Reviewer #2: Partly

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: No

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Reviewer #1: In this manuscript, the authors revealed the effects of Debaryomyces hansenii on diversity of bacterial lactase genes from intestinal mucosa of antibiotic-associated diarrhea (AAD) mice. They established diarrhea model, extracted genomic DNA of intestinal mucosa, amplified β-galactosidase gene by PCR and sequenced them using high-throughput sequencing technology. This is an interesting manuscript, generally carefully prepared, but it still requires significant revision before publication. In particular, it is lacking in essential details and information.

1.The antibiotic-associated diarrhea (AAD) mice model is the core and principal content of this manuscript. However, there is no evidence that can comfirm the model is sucsessfully established.Please provide the data.

2. As the authors mentioned “There are about 1000 species of bacteria inhabiting in human’s intestinal tract”, while they checked the β-galactosidase gene only in Lactobacillus and Escherichia coli. Why choose these two? Can the β-galactosidase genes in Lactobacillus and Escherichia coli represent that in all intestinal microorganisms?

3. Eighteen mice, which were randomly divided into three groups (6 mice per group), healthy control group, diarrhea model group and D. hansenii treatment group, were used in the present study. However, they used samples from nine mice for β-galactosidase gene amplification and sequencing. Why not check the β-galactosidase gene in all eighteen mice? Do you think the data from three sample per group is enough for clarification the influence of Debaryomyces hansenii on bacteria lactase gene diversity in intestinal mucosa of mice with antibiotic-associated diarrhea?

4. The writing need to be improved.

Reviewer #2: The submitted manuscript pertains to the influence of Debaryomyces hansenii on bacteria lactase gene diversity in mucosa of mice with antibiotics-associated diarrhea. The starting point of the study is the statement that antibiotic – associated diarrhea may be related to the decreasing activity of lactase. The aim of the work is clearly defined and has a practical perspective - to determine whether D. hansenii could be used in the treatment of antibiotic-associated diarrhea due to its effect on the regulation of lactase gene expression. The manuscript is concise, the abstract adequately describe the experimental question and results and the title is appropriate. The experimental results support the conclusions. I believe that the paper can be published once the authors address the issues below.

1) Throughout the manuscript, the use of the English language needs to be drastically improved. Numerous grammatical errors are present in the manuscript. This is a major concern that must be addressed before publication.

2) More references are appropriate, such as for the experimental techniques. For example, what is the source reference for the universal primers according to lactase gene sequences of Lactobacillus and Escherichia coli ?

3) The author should provide the data about the fecal consistency, fecal water content and stool weight after treatment with antibiotics or D. hansenii ?

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2019 Dec 6;14(12):e0225802. doi: 10.1371/journal.pone.0225802.r002

Author response to Decision Letter 0

12 Sep 2019

Dear editor and reviewers:

Firstly, thanks reviewers and editor for their valuable comments on our manuscript entitled " Influence of Debaryomyces hansenii on bacteria lactase gene diversity in intestinal mucosa of mice with antibiotic-associated diarrhea" (PONE-D-19-19505). Those comments are very helpful for revising and improving our paper, as well as the important guiding significance to other research. We have studied the comments carefully and made corrections which we hope meet with approval. The main corrections are in the manuscript and the response to the reviewers’ comments are as follows:

Replies to the reviewers’ comments:

Reviewer #1:

Response: The antibiotic-associated diarrhea (AAD) first established by Ao Zeng, on the basis of which our research group has done a lot of researches, including the diversity of intestinal contents and intestinal mucosal bacteria, and the diversity of intestinal contents and intestinal mucosal lactase gene, fully demonstrating the reliability of this model.

1. Zeng A, Zhang HL, Tan ZJ, et al. The construction of mice diarrhea model due to dysbacteriosis and curative effect of ultra-micro Qiweibaizhusan. Microbiology China. 2012; 39(9): 1341-1348. https://doi.org/ 10.13344/j.microbiol.china.2012.09.012

2.Long C, Liu Y, He L, et al. Bacterial lactase genes diversity in intestinal mucosa of mice with dysbacterial diarrhea induced by antibiotics. 3 Biotech. 2018; 8(3): 176. https://doi.org/ 10.1007/s13205-018-1191-5 PMID: 29556430

3. Long CX, He L, Guo YF, et al. Diversity of bacterial lactase genes in intestinal contents of mice with antibiotics-induced diarrhea. World Journal of Gastroenterology. 2017; 23(42):7584-7593. https://doi.org/ 10.3748/wjg.v23.i42.7584 PMID: 29204058

2.As the authors mentioned “There are about 1000 species of bacteria inhabiting in human’s intestinal tract”, while they checked the β-galactosidase gene only in Lactobacillus and Escherichia coli. Why choose these two? Can the β-galactosidase genes in Lactobacillus and Escherichia coli represent that in all intestinal microorganisms?

Response: There are few lactase gene sequences published in NCBI database. We downloaded published 89 bacterial lactase gene sequences from NCBI database, and the sequence sources of these genes include Bifidobacterium, Lactobacillus, Enterobacter, Streptococcus, Bacillus, Micrococcus and Clostridium. According to these bacterial lactase gene sequences, we designed 7 pairs of universal primers for the analysis of bacterial lactase gene diversity. After PCR amplification and metagenomic sequencing, it was found that universal primers of bacterial lactase gene from Lactobacillus and Escherichia coli had good specificity, high sensitivity and could annotate more microbial species.

1.Long CX, He L, Liu YJ, et al. Universal primer for analysis of the diversity of intestinal bacterial lactase gene. Chinese Journal of Applied and Environmental Biology. 2017, 23(04): 758-763. https://doi.org/ 10.3724/SP.J.1145.2016.10008

Response: Admittedly, the bioduplication of the three samples in each group is a little low, but it can meet the minimum requirements of statistics. In our experiment, there were 6 mice in each group. When collecting intestinal mucosa, we randomly mixed the mucosa of a male and a female mouse in the group. On the one hand, the intestinal mucosa of mice was small, and we were worried that the extracted DNA was not pure enough. On the other hand, there were some differences between the intestinal flora of male mice and female mice. In order to fully reflect the characteristics of the mucosal bacteria of mice, the intestinal mucosa of a male mouse and a female mouse were mixed together.

4. The writing need to be improved.

Response: Thanks for the expert's advice. We will check the manuscript carefully to improve the readability of the article.

Reviewer #2:

1.Throughout the manuscript, the use of the English language needs to be drastically improved. Numerous grammatical errors are present in the manuscript. This is a major concern that must be addressed before publication.

Response: Thanks to the expert's valuable advice, we will ask a native English speaker to polish our manuscript.

2.More references are appropriate, such as for the experimental techniques. For example, what is the source reference for the universal primers according to lactase gene sequences of Lactobacillus and Escherichia coli ?

Response: The sources of the universal primers for the analysis of lactase gene diversity were designed by our research group. According to the lactase gene sequence registered in NCBI database, DANMAN software was used to find the conservative region of the lactase gene sequence, and the Primer Premier 5.0 software was used to design upstream and downstream primers on both ends of conserved regions. Finally, the synthesis of primers was completed by Shanghai pessenol biotechnology co., LTD.

1.Long CX, He L, Liu YJ, et al. Universal primer for analysis of the diversity of intestinal bacterial lactase gene. Chinese Journal of Applied and Environmental Biology. 2017, 23(4): 758-763. https://doi.org/ 10.3724/SP.J.1145.2016.10008

2.The author should provide the data about the fecal consistency, fecal water content and stool weight after treatment with antibiotics or D. hansenii ?

Response: In this study, we did not conduct quantitative analysis of fecal concentration, fecal water content and fecal weight after treatment with antibiotics or D. hansenii, but in previous articles, we have qualitatively recorded the wetness of stool after antibiotic modeling and yeast treatment in mice.

1.Guo KX, Tan ZJ, Xie MZ, et al. The synergic effect of ultra-micro powder Qiweibaizhusan combined with yeast on dysbacteriotic diarrhea mice. Chinese Journal of Applied and Environmental Biology. 2015; 21(1): 61-67. https://doi.org/ 10.3724/sp.j.1145.2013.10002

Replies to the editors’ suggestions

1.You should revise your language with a native English speaker.

Response: Thanks for the editor's advice, in terms of language quality, we invited our colleagues working in the United States to polish the manuscript to make it more accurate.

2.You should upload your raw data into public database such as NCBI and provide the accession number. Table 1 should be deleted as the data could be gained according to your raw data.

Response: As required, we have uploaded all the original sequences to NCBI database. The accession number is SRP220043 (https://www.ncbi.nlm.nih.gov/Traces/sra_sub/), which was released on September 6, 2019. According some information on the Internet, the data uploaded to NCBI database might not be retrieved for 3 months. So I listed my NCBI database(https://www.ncbi.nlm.nih.gov/) account and password at the end. Account number: heyunshan; password: 20183371he

3.For data analysis, you should perform data normalization, i.e. each sample with same read numbers, before the subsequent data analysis.

Response: Thanks to the editors for their advice, the sample size of each group is consistent in any one data analysis. The data analysis of this experiment was conducted by professionals of pessenol biological company, and the data were authentic and reliable with strong comparability.

4.Alpha diversity analysis should included richness indices and diversity indices. Please added these indices.

Response: Regarding Alpha diversity, Simpson index was added, which could well reflect community diversity.

5.For PCoA analysis, you should include different algorithms such as weighted UniFrac, unweighted UniFrac and Bray-Curtis.

Response: For beta analysis, PCA and NMDS analysis are selected instead of PCoA analysis. NMDS analysis is not affected by the numerical value of sample distance, and only considers the size relationship between them. For data with complex structure, NMDS analysis ranking results may be more stable.

6.For differential bacteria analysis, you should perform LEfSe analysis.

Response: In the differential bacteria analysis, we introduced LEfS analysis as required. LEfSe analysis show that Sphingomonadaceae, Sphingomonas and Sphingomonadales were the key colony members in D. hansenii treatment group, while no key colony members were found in healthy control group and diarrhea model group.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(19.9KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0225802.r003

Decision Letter 1

Zongxin Ling

8 Oct 2019

PONE-D-19-19505R1

Influence of Debaryomyces hansenii on bacteria lactase gene diversity in intestinal mucosa of mice with antibiotic-associated diarrhea

PLOS ONE

Dear Professor Tan,

From your response, we could not find the significant improve of the manuscript especially the data analysis. You said that you had performed data normalization, which was important for the data interpretation. However, I could not find any information about the data normalization. How many reads had been normalized in your present study? from your raw data, I found that there were significantly different in the read numbers in each samples. From your Table 1 in your original submission, you could find them more clearly. From your rarefaction and ran abundance curve, I thought that you did not perform data normalization at all. I insisted that you should re-perform your data analysis before I could consider this manuscript again.

We would appreciate receiving your revised manuscript by Nov 22 2019 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Zongxin Ling

Academic Editor

PLOS ONE

PLoS One. 2019 Dec 6;14(12):e0225802. doi: 10.1371/journal.pone.0225802.r004

Author response to Decision Letter 1

6 Nov 2019

Dear Editors:

Due to the negligence of the company, there are some problems with the raw data of tlmm1 sample. As a result, when you carefully examine the raw data, you find that there were significantly different in the read numbers in each samples. After communication with the company, the raw data of tlmm1 sample has been recovered from the company's database. I want to upload all the original data to NCBI database again, but due to duplication with the previously uploaded data, so it has been unable to pass the review of NCBI database. Therefore, the raw data will be sent to PLOS ONE's submission system in the form of attachment. Data analysis has asked the company to analyze the original data according to the latest industry standards, and the analysis results are basically the same as before. The language of the article has been requested to be retouched by AJE company, and the retouching certificate has been uploaded in the form of attachment. Thank you again for taking time out of your busy schedule to review the manuscript.

Best regards.

Yours sincerely,

Yunshan He

Correspondence author:

Name: Wenge Li; Zhoujin Tan

E-mail: 641386565@qq.com; tanzhjin@sohu.com

PLoS One. doi: 10.1371/journal.pone.0225802.r005

Decision Letter 2

Zongxin Ling

13 Nov 2019

Influence of Debaryomyces hansenii on bacteria lactase gene diversity in intestinal mucosa of mice with antibiotic-associated diarrhea

PONE-D-19-19505R2

Dear Dr. Tan,

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

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. 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.

With kind regards,

Zongxin Ling

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0225802.r006

Acceptance letter

Zongxin Ling

20 Nov 2019

PONE-D-19-19505R2

Influence of Debaryomyces hansenii on bacterial lactase gene diversity in intestinal mucosa of mice with antibiotic-associated diarrhea

Dear Dr. Tan:

I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Zongxin Ling

Academic Editor

PLOS ONE

Associated Data

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

Supplementary Materials

S1 Dataset. The raw data of healthy control group (tlcm).

(ZIP)

Click here for additional data file.^{(40.2MB, zip)}

S2 Dataset. The raw data of diarrhea model group (tlmm).

(ZIP)

Click here for additional data file.^{(50.2MB, zip)}

S3 Dataset. The raw data of D. hansenii treatment group (tljm).

(ZIP)

Click here for additional data file.^{(24.8MB, zip)}

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(19.9KB, docx)}

Data Availability Statement

All relevant data are within the manuscript.

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PERMALINK

Influence of Debaryomyces hansenii on bacterial lactase gene diversity in intestinal mucosa of mice with antibiotic-associated diarrhea

Yunshan He

Yuan Tang

Maijiao Peng

Guozhen Xie

Wenge Li

Zhoujin Tan

Roles

Abstract

Aim

Methods

Results

Conclusion

Introduction

Materials and methods

Reagents and medicine

Animals and procedures

Total DNA extraction from intestinal mucosa

PCR amplification of the mucosal bacterial lactase gene and high-throughput sequencing

Gene diversity and statistical analysis

Results

Sequence statistics and OTU analysis

Fig 1. OTUs of the bacterial lactase genes from intestinal mucosa.

Alpha diversity of the bacterial lactase gene from the intestinal mucosa of AAD mice treated with D. hansenii

Fig 2. Rarefaction curve of the bacterial lactase gene from the intestinal mucosa in each sample.

Fig 3. Rank abundance curve of each sample.

Table 1. Alpha diversity indices of the bacterial lactase gene from the intestinal mucosa of antibiotic-associated diarrhea mice treated with D. hansenii.

Beta diversity of bacterial lactase genes in the intestinal mucosa of AAD diarrhea mice treated with D. hansenii

Fig 4. PCA analysis of the bacterial lactase gene from intestinal mucosa.

Fig 5. NMDS analysis of the bacterial lactase gene from intestinal mucosa.

Abundance and source of the bacterial lactase gene from the intestinal mucosa of AAD mice treated with D. hansenii

Fig 6.

Fig 7. Source of lactase-producing bacteria from intestinal mucosa.

Discussion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Zongxin Ling

Roles

Author response to Decision Letter 0

Decision Letter 1

Zongxin Ling

Roles

Author response to Decision Letter 1

Decision Letter 2

Zongxin Ling

Roles

Acceptance letter

Zongxin Ling

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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