Isolation, identification, and biological characteristics of Clostridium sartagoforme from rabbit.

In order to develop microbial additives for rabbit feed, a spore-forming bacteria was isolated from the feces of Hyla rabbit using reinforced clostridium medium (RCM). The 16S rDNA sequence of the bacterium was subjected to pairwise sequence alignment using BLAST; the colony morphology, and physiological, biochemical, and stress resistance were studied. The results showed that the bacterium was Clostridium sartagoforme, a gram positive anaerobe, which can produce spores. The colony diameter was 0.5 mm-2.5 mm, the diameter of the bacteria was 0.5 μm-1.0 μm × 2.0 μm-6.3 μm, and the spore diameter was 1 μm-1.2 μm × 1 μm-1.2 μm. C. sartagoforme can utilize various sugars and alcohols such as fructose, galactose, sorbitol, and inositol. It secreted cellulase into the extracellular environment to form a transparent hydrolysis circle in Congo red medium, it could not liquify gelatin, and the lysine decarboxylase reaction was positive. In liquid medium it entered the stable growth period after 9 h of inoculation. Additionally, it had good stress resistance with a survival rate that exceeded 53% after gastric juice (pH 2.5) treatment for 3 h, it grew in a medium with a bile salt concentration of 0.3%, and the survival rate exceeded 85% after 10 minutes at 80°C. Moreover, animal testing indicated that this strain has no adverse effects on the morbidity and mortality of rabbits. In summary, C. sartagoforme XN-T4 was isolated from rabbit feces. This bacterium has good resistance to stress, can decompose a variety of monosaccharides and polysaccharides including cellulose, which is relatively harmless for animal health.

Introduction

Rabbits are herbivores, there are abundant cellulose degrading bacteria resources in rabbit intestinal tract, because they rely on the microorganisms in the caecum to digest cellulose but not produce cellulase by themselves [1]. In addition, cellulose degrading bacteria isolated from animals have natural advantages in the utilization of crude fiber feed. Most of them are inherent bacteria in the intestinal tract, which are easier to adapt to the intestinal environment of the animal and play a role [2].

Clostridium sartagoforme is an anaerobic spore producing bacterium [3, 4], which determines that the bacterium has a natural advantage in resisting the effects of digestive juice and adapting to the anaerobic environment of the gastrointestinal tract [5]. Researches have shown that the strain can decompose microcrystalline cellulose, carboxymethyl cellulose and chitin [4, 6, 7], and its metabolites include formic acid, acetic acid, propionic acid, lactic acid, butyric acid, isovaleric acid and hydrogen [4, 8, 9]. Simůnek J et al. isolated C. sartagoforme from feces of horse, and this strain has high exocellular activity of N-Acetylglucosaminidase [6]. Zhang JN isolated Clostridium sartagoforme FZ11 from cow dung compost, using urea as a nitrogen source and raw corn stalk as a substrate, confirming that this strain could decompose cellulose and have good hydrogen production characteristics [7]. In the research of Nathani Neelam M and and his coleagues, Clostridium sartagoforme AAU1 was isolated from Surti buffalo rumen liquor, and they pointed out that VFAs produced by the bacteria play an important role in animal health maintenance through genomic analysis [10]. At present, the research about C. sartagoforme isolated from digestive tract or feces of rabbits has not been reported, and there is little information on the biological characteristics of this strain. In this experiment, C. sartagoforme was isolated and identified from rabbit feces, and its physiological, biochemical and stress resistance characteristics were preliminarily studied, which help to provide reference for the further development of rabbit microbial additives.

Materials and methods

Samples

Fresh fecal samples were taken from Hyla, Rex, and Hycole rabbits at Tongteng Biotechnology Co., Ltd., Yangling Tianxin Rabbit Industry Co., Ltd., and Baoji Enyi Rabbit Farm, respectively. Samples were put into individual sterilized tubes, placed in liquid nitrogen, and rapidly returned to the laboratory.

Media

Reinforced Clostridium Medium (RCM) contained the following ingredients: peptone (10.00 g), beef extract (10.00 g), glucose (5.00 g), sodium chloride (5.00 g), yeast powder (3.00 g), sodium acetate (3.00 g), soluble starch (1.00 g), L-cysteine hydrochloride (0.50 g), agar (20.00 g) (added in solid medium), and distilled water to bring the final volume of the solution to 1.00 L.

Congo red-sodium carboxymethyl cellulose medium (CMC) was prepared with Congo Red (0.20 g), KH2PO4 (2.00 g), MgSO4 (0.15 g), agar (15.00 g), yeast powder (2.00 g), sodium carboxymethyl cellulose (10.00 g), and distilled water to bring the solution to a final volume of 1.00 L.

Isolation and culture methods

A 10 g sample of the collected rabbit feces was placed in a sterilized beaker with 90 mL PBS. The beaker was placed in a 80°C water bath for 10 minutes to kill the non-spore bacteria; the sample was transferred to 400 mL of RCM liquid medium, sealed with sterilized liquid paraffin, and cultured at 37°C for 36 h. After culture, the concentrated bacterial solution was diluted to 10−3, 10−5, 10−7. The diluted solutions were applied to the RCM solid medium, the petri dishes were placed into a sterilized container that could be tightly sealed, oxygen was removed by excessive pyrogallic acid, and the dishes were cultured at 37°C for 48 h. The consumption of pyrogallic acid for deaeration was calculated according to the following chemical reaction equation:

After culture, according to the colony morphology of C. sartagoforme, colonies were selected for microscopic examination; the colonies that met the characteristics continued to be anaerobic cultured with RCM solid medium (37°C, 48 h). This step was repeated until a monoclonal colony was obtained.

Identification of C. Sartagoforme

Morphological identification

Identification was performed according to the colony morphology and Gram staining characteristics of the monoclonal colonies. Additionally, the culture time of the medium used for Gram staining was controlled at around 24 h. Clostridium is generally positive for Gram staining, but the cell wall structure of the old bacteria would become loose if the culture time is too long, and the result of Gram negative may appear after staining [11].

16S rDNA gene sequence analysis and identification of target strain

The target strains’ DNA was extracted by a bacteria genomic DNA extraction kit (Takara Bio Inc., Shiga, Japan). The quality of genomic DNA was determined by 1% agarose gel electrophoresis. The concentration and purity of DNA were determined by ultra-micro nucleic acid analyzer. PCR amplification was carried out according to the required DNA samples. The following 16S r DNA PCR universal primers were used for amplification: 7F- CAGAGTTTGATCCTGGCT and 1540R- AGGAGGTGATCCAGCCGCA. The PCR conditions are shown in Table 1.

Table 1

C. sartagoforme 16S rDNA PCR reaction system.

Materials	Volume
2 × Mix	7.5 μL
Template DNA	0.5 μL
Forward Primer	0.5 μL
Reverse Primer	0.5 μL
DD H2O	6 μL
Total Volume	15 μL

Note: 2 × Mix (Purchased from ComWin Biotech Co.,Ltd., Beijing).

PCR fragments were synthesized at the following time-temperature regimes: initial denaturation at 94°C for 5 min; subsequent 34 cycles at 94°C for 30 s, at 58°C for 30 s, and at 72°C for 30 s; final polymerization at 72°C for 2 min.

The PCR products were detected by 1% agarose gel electrophoresis, and the products with a fragment of about 1,500 bp were sent to Beijing AUGCT Company for sequencing. The sequencing results were run through NCBI for BLAST homology comparison and a phylogenetic tree was constructed; the comparison results were combined with the phylogenetic tree to determine the strain species.

Physiological and biochemical characteristics of C. sartargoforme

Gelatin liquefaction and sugar fermentation characteristics

The physiological and biochemical characteristics of the target strain were studied by micro-biochemical reaction identification tube for bacteria (HOPEBIO, Qingdao, Shandong). The bacteria were inoculated in the clean bench, and the biochemical reaction tube was liquid sealed with sterilized liquid paraffin and covered with parafilm.

Fiber decomposition characteristics

The target strain was inoculated on CMC-Congo Red plate for anaerobic culture, and the fiber degradation ability of the strain was determined by the fiber hydrolysis circle on the medium.

Growth and pH curves

In order to generate the growth and pH curves, 96 tubes containing 10 mL RCM liquid medium, 72 of which were inoculated with target strain at 1% of inoculation volume, were cultured in anaerobic conditions at 37°C. Three tubes were taken out every 1 h during this period. Three tubes with inoculated bacteria and one uninoculated tube (blank reference) were paired to measure the OD600 and pH. Based on the data, growth and pH curves were generated.

Heat resistance characteristics

Under aseptic condition, the target bacterial solution after 24 h of culturing was put into four sterilized centrifuge tubes; the tubes were placed at room temperature, and respectively bathing in 80°C, 90°C, and 100°C water for 10 min. Following the exposure to various temperatures, the bacteria were cultured in solid RCM for 48 h and then counted. Taking the number of colonies in the room temperature treatment group as 100%, the ratio of other groups to it is calculated as the survival rate.

Gastric acid and bile salt tolerance

The pH of artificial gastric juice was adjusted to 1.5, 2.5, and 3.5 with 5 M HCl [12]. 10 mL fresh culture was centrifuged at 3,000 rpm for 10 min, then the pelleted cells were suspended in 10 mL pH 1.5, 2.5, and 3.5 artificial gastric juice and incubated in 37°C for 1 h, 2 h, and 3 h respectively. After incubation, samples were washed three times with phosphate-buffered saline (PBS; pH 7.2) before serially diluted up to 10−8, 50 μL of the diluted cultures was spread on RCM agar plates and incubated for 48 h at 37°C, 6 replicates for each sample, followed by colony counting. The survival rate (%) in gastric acid was calculated with the mean of colony counts of the plates after treatment with respect to their mean before treatment. The results were presented in mean number of surviving bacteria ± standard deviation [13].

In the bile salt tolerance study, the concentrations of bovine bile salt in liquid medium were 1.0 g/L, 1.5 g/L, 2.0 g/L, 2.5 g/L, 3.0 g/L, 3.5 g/L, and 4.0 g/L. After sterilization at 120°C for 20 min, the target strain was inoculated according to 1% inoculation volume. Sealing with liquid paraffin, cultured at 37°C for 24 h before the OD600 was measured.

Safety test

Animals

A total of 162 35-day-old Rex rabbits (half males and half females) were randomly divided into 3 groups with 9 replicates per group and 6 rabbits per replicate. Rabbits were housed in cages (1 female and 1 male rabbit/cage) and the trial lasted 35 days. This test was carried out in Weinan New Rex rabbit Farm, Weinan (China).

Clostridium sartagoforme was added into drinking water. A, B and C respectively represent the amount of bacteria added according to the body weight of Rex rabbits: 108 CFU/Kg, 106 CFU/Kg and medium mixture. Adding amount was based on the related studies of Clostridium butyricum [14].

The diarrhea rate and mortality were tested. Diarrhea rate: the number of diarrhea per day in each treatment group was recorded (diarrhea, too large or too small feces and jelly-like feces were regarded as diarrhea), and the diarrhea rate of the experimental rabbits was calculated by the following calculation method [15]:

Ethic statement and animal experiments permission

The protocol was approved by the Committee on the Ethics of Laboratory Animals of Northwest Agriculture & Forest University (approved number: 0369/2018).

Statistical analysis

The experimental data were analysed using SPSS 22.0 software (SPSS Inc., Chicago, IL, USA), and one-way analysis of variance were used to examine the gastric acid tolerance of Clastridium sartagoforme, mortality and diarrhea rate of rabbits. The results were expressed as mean ± SEM. A statistical significance was declared at P < 0.05 and extremely significant were defined as P < 0.01.

Results

Isolation and identification of C. sartagoforme

According to the morphology results, Gram staining, and microscopic examination of the cultured strains, nine eligible strains were obtained. Using the DNA of each strain as a template, the 16S rDNA was amplified using universal primers, and the amplified products were electrophoresed on a 1% agarose gel. The electrophoresis results of the target strain XN-T4 are shown in Fig 1. PCR for the strains and the amplified products were sequenced to identify their specific species. The results are shown in Table 2.

Fig 1

Agarose gel electrophoresis of C. sartagoforme XN-T4 16S rDNA.

Table 2

Identification of the isolated bacteria.

Strain Number	Strain identified
XN-T14	Clostridium sordellii
XN-D1	Clostridium tertium
XN-D2	Clostridium tertium
XN-D3	Clostridium tertium
XN-T4	Clostridium sartagoforme
XN-D5	Clostridium tertium
XN-B2	Clostridium sordellii
XN-B3	Clostridium tertium
XN-B5	Clostridium tertium

The target strain XN-T4 16S rDNA was sequenced with a length of 1,432 bp and the accession number is MW450913.

Pairwise sequence alignment of the sequence to BLAST showed that the strain XN-T4 was closely related to C. sartagoforme (99% homology over a 100% sequence query cover). Several strains with higher homology were selected to make a phylogenetic tree. The sequences were aligned using MEGA (6). Phylogenetic analysis using maximum likelihood. The results are shown in Fig 2. Therefore, XN-T4 could be considered C. sartagoforme.

Fig 2

Phylogenetic tree of C. sartagoforme XN-T4 16S rDNA.

Morphological characteristics

C. sartagoforme XN-T4 was cultured on solid RCM for 24 h. The morphology is shown in Fig 3. The colony was white, round with neat edges, moist, smooth, and opaque, with a diameter of 0.5 mm-2.5 mm. Gram staining was positive, bacteria occurred as single cells or in pairs, the spores were terminal, the bacteria were drumstick-shaped when spores were formed, the diameter of the bacteria was 0.5 μm-1.0 μm × 2.0 μm-6.3 μm, and the spore diameter was 1 μm-1.2 μm × 1 μm-1.2 μm.

Fig 3

Colonial and cell morphology of C. sartagoforme XN-T4.

Physiological and biochemical characteristics

C. sartagoforme XN-T4 could decompose glucose, sucrose, and galactose, but not arabinose. It could not use inositol or dulcitol, it did not liquefy gelatin, and the reaction of lysine decarboxylase was positive. These results are shown in Table 3.

Table 3

Identification of the physiological characteristics of C. sartagoforme XN-T4.

Items	Results	Items	Results
Arabinose	−	Mannitol	+
Mannose	+	Sorbitol	+
Glucose	+	Inositol	−
Galactose	+	Dulcitol	−
Fructose	+	Lysine decarboxylase	+
Rhamnose	+	Gelatin hydrolysis	−
Sucrose	+

Notes: + indicates that the result is positive, − indicates that the result is negative.

Fiber hydrolysis characteristics

C. sartagoforme XN-T4 produced clear and visible hydrolysis circles on CMC-Congo Red plate (Fig 4), ratio of the hydrolytic circle and the strains colony is approximately 2.5. This indicating that the bacteria could decompose and utilize cellulose and secrete cellulase outside of the cell.

Fig 4

Characteristic of C. sartagoforme XN-T4 on Congo-CMC.

Growth characteristics

The growth curve of C. sartagoforme XN-T4 is shown in Fig 5. The lag phase was about 5 h after inoculation, and then it entered the log phase, which lasted about 5 h before entering the stable phase. The pH also started to drop rapidly at 5 h; this drop lasted for about 10 h after which the pH stabilized at about 4.9.

Fig 5

Growth and pH curve of C. sartagoforme XN-T4.

Thermal tolerance

C. Sartagoforme XN-T4 had good heat resistance. The survival rate exceeded 85% after treatment at 80°C for 10 min, but the survival rate dropped rapidly to less than 10% after exposure to 90°C. All strains died after treatment at 100°C (Table 4).

Table 4

Survival rate of C. sartagoforme XN-T4 at different temperatures after 10 min.

	Temperature
	80°C	90°C	100°C
Survival rate (%)	85.55±8.82	8.06±1.70	0

Gastric acid and bile salt tolerance

The results showed that the survival rate of C. sartagoforme XN-T4 treated at pH 1.5 for 1 h was more than half, and that for 3 h under pH 2.5 and pH 3.5 was more than 50%, as shown in Table 5.

Table 5

Survival rate of C. sartagoforme XN-T4 in artificial gastric juice.

Conditions		Hours
		1	2	3
Survival rate (%)	pH = 1.5	58.65±4.22a	37.54±4.29b	31.09±7.34b
	pH = 2.5	79.95±2.32A	51.56±2.26B	53.39±3.76B
	pH = 3.5	88.97±0.83A	74.75±1.39B	58.33±1.54C

Notes: Different lowercase letters on the upper right shoulder of the same row indicate significant differences (P < 0.05), and uppercase letters indicate extremely significant differences (P < 0.01).

C. sartagoforme XN-T4 had good bile salt tolerance. It grew well in the liquid medium with 0.15% bile salt content. It still grew in the liquid medium with 0.3% bile salt content, but could not grow in the medium with 0.35% or more bile salt content (Table 6).

Table 6

C. sartagoforme XN-T4 bile salt tolerance.

Content of bile salt	0.10%	0.15%	0.2%	0.25%	0.3%	0.35%	0.4%
OD 600	1.01±0.17	1.01±0.20	0.33±0.16	0.27±0.11	0.11±0.08	0	0

There were no significant differences in diarrhea rate and mortality among groups A,B and C, as shown in Table 7.

Table 7

Effects of C. sartagoforme XN-T4 on the diarrhea rate and mortality of Rex rabbits (%).

	A	B	C
Diarrhea rate	0.69±0.18a	0.85±0.09a	0.74±0.33a
Mortality	1.85±5.6a	1.85±5.6a	1.85±5.6a

Notes: A, B and C respectively represent the amount of bacteria added according to the body weight of Rex rabbits: 108 CFU/Kg, 106 CFU/Kg and medium mixture. Different lowercase letters on the upper right shoulder of the same row indicate significant differences (P < 0.05).

Discussion

Isolation and identification of C. sartagoforme XN-T4

In this experiment, C. sartagoforme was only isolated from Hyla rabbit, but not from Rex or Hycole rabbits. This is likely due to the species of rabbit, age, and food ingredients in the region [16]. Bogatyrev et al. pointed out that the composition of the host’s intestinal flora depends on the host’s species, diets, age, and gender [17].

C. sartagoforme can produce spores. Accordingly, the sample was heated and pretreated to kill the non-spore bacteria, and the target strain was isolated by repeated streaking inoculation and purification. According to the colony morphology and Gram staining characteristics, the cultured strains were selected. Combined with sugar and alcohol fermentation experiments, gelatin liquefaction experiments and molecular identification, the strain was identified as C. sartagoforme [18].

16S rDNA is the gene of the 16S rRNA subunit of bacterial ribosomes. As the most commonly used molecular identifier in bacterial taxonomy, it is about 1,500 bp. It not only reflects the differences between different bacterial genera, but also facilitates sequence determination [19]. This study directly performed 16S rDNA sequence determination on the selected strains on the basis of the selection of colony morphology and strain Gram staining characteristics. Gelatin liquefaction and sugar fermentation tests were performed as auxiliary identification, which effectively reduced the workload and the test cost, and the target strain was accurately identified.

Physiological and biochemical characteristics of C. sartagoforme XN-T4

Bacterial spores have thick spore walls, multiple spore membranes, a solid structure, low water content (80% of propagules and 40% of spores), an almost stopped metabolism, and a strong refractive index [20]. Because the spores produce a unique dipicolinic acid, combined with calcium, the spores have a strong resistance to adverse physical and chemical environmental conditions; they are especially resistant to high temperature, drying and osmotic pressure, and ordinary chemicals do not easily penetrate them [20]. The C. sartagoforme XN-T4 isolated in this experiment can form spores to resist adverse environmental conditions, which is beneficial to the production and preservation of the strain.

C. sartagoforme can use a variety of sugars and alcohols as carbon sources. The strain isolated in this experiment could decompose mannitol, but could not use arabinose or inositol. This is consistent with the results of the API bacterial identification system. The C. sartagoforme isolated from the rumen of buffalo is contrary to the results of this experiment [10], which may be due to the fact that many physiological and biochemical characteristics of bacteria are encoded by extrachromosomal genetic factors. The factors that affect the expression of physiological and biochemical characteristics are more complicated [12]; therefore, the results were different. Clostridium sartagoforme XN-T4 can liquefy gelatin, which is consistent with the previous reports [21, 22].

Although rabbits are herbivores, they cannot secrete cellulase themselves, they mainly rely on cecal symbiotic microorganisms to decompose cellulose in the feed [1]. These microorganisms decompose cellulose and produce volatile fatty acids(VFA) for rabbits to use [23]. In addition, due to the coprophagy of rabbits, microorganisms can provide certain bacterial proteins for domestic rabbits [24]. The transparent circle on the CMC-Congo Red plate indicates that the bacterium can decompose cellulose, it means that C. sartagoforme XN-T4 has the potential to improve the digestibility of cellulose for herbivores as a microbial feed additive.

Safety, growth characteristics and stress resistance of C. sartagoforme XN-T4

There is no report that C. sartagoforme is a pathogen, and the data of C. sartagoforme XN-T4 in animal feeding experiments shows that this strain has no adverse effects on the health of rabbits. This demonstrates that the strain is safe to a certain extent. In addition, the composition of primary metabolites and secondary metabolites should be further studied to confirm its security.

The growth of bacteria can be divided into the delay phase, logarithmic phase, constant phase, and decay phase [25]. The number of newly propagated cells and the number of dead cells in the constant phase are basically in a stable state, and the total number of viable bacteria is at the highest level. This stage is the best time to obtain bacteria [26]. C. sartagoforme XN-T4 began to enter the logarithmic phase about 5 h after inoculation, the logarithmic phase lasted for about 5 h, and then entered the constant phase. Through the growth curve of C. sartagoforme XN-T4, it is possible to know which growth period the bacteria are in. In the preparation of microbial additives, the timing of termination of culture can be accurately selected to maximize the yield of bacteria.

In the growth curve of C. sartagoforme XN-T4, after entering the logarithmic phase, the pH of the fermentation broth continued to decrease, indicating that volatile fatty acids were produced during the growth of the bacteria [25]. A study showed that the VFA produced during the growth of C. sartagoforme are mainly acetae, formiate, butyrate and lactate [8]. VFA accounts for 40% of rabbit maintenance energy requirement [27].

C. sartagoforme XN-T4 could tolerate 80°C for 10 min and ensured that the number of viable bacteria was not less than 80%, indicating that the strain had certain heat resistance. However, as the temperature reached 90°C, the number of viable bacteria decreased rapidly. Microbial additives can be added by mixing in feed or drinking water. Because rabbits do not like to eat powder or wet feed and have rodent behavior, pellet feed is usually used. The temperature of pellet feed in the circular mould pelleting machine is often above 80°C. Therefore, if C. sartagoforme XN-T4 is used as the microbial additive, the method of adding drinking water is more appropriate.

Gastric juice in the stomach and bile salt in the small intestine can kill the vast majority of bacteria in food and protect the health of the body. In resting gastric glands, the intragastric pH is approximately 3–4 [28, 29], and gastric secretion is neither influenced by food intake nor fasting [30]. The strain can not only survive in the medium with bile salt concentration of 0.3%, but also maintain a certain degree of growth, indicating that the bacterium has good tolerance to bile salt, which is similar to the results of Wang TH on Clostridium butyricum [15], which belongs to the same genus of Clostridium. When microbial additives are used in monogastric animals such as rabbits, it is necessary to ensure that a sufficient number of strains reach the cecum before their beneficial effects can be achieved. C. sartagoforme XN-T4 has good tolerance to gastric juice and bile salt. The survival rate of C. sartagoforme XN-T4 treated at pH 3.5 for 3 h was more than half, and it could grow in the medium with bile salt concentration of 0.3%. This indicated that the strain could tolerate the action of gastric juice and bile salt and reach the hindgut.

Conclusion

A strain of Clostridium sartagoforme XN-T4 was isolated from Hyla rabbit feces by RCM medium. Through colony characteristics and cell morphology, combined with physiological and biochemical methods and PCR identification. The strain has good stress resistance, can decompose cellulose, and has no obvious toxic effect on rabbits. Theoretically, this bacteria has a strong ability to resist the effects of gastric acid and bile salts in animal digestive juices. It may be a candidate for microbial additives to improve the digestibility of cellulose in rabbits. Futher more, for its secondary metabolite composition, safety and in vitro and in vivo metabolic characteristics still need experimental research.

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27 Oct 2020

PONE-D-20-28305

Isolation, identification and biological characteristics of Clostridium sartagoforme from rabbit

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Reviewer #1: This manuscript reports the isolation, identification and biological characteristics of Clostridium sartagoforme from rabbit. The study is original. However, it is necessary to add more data to make the study solid from the scientific viewpoints. In particular, the authors should add more research data on the probiotic effect and safety of Clostridium sartagoforme in vitro. In addition, the following points need to be revised and clarified.

1. English usage should be checked thoroughly by native users. There is still room to improve the language and the grammatical errors in the manuscript need to be corrected.

2. In Abstract, “a bacillus-producing strain was isolated from the feces of Hyla rabbit using reinforced clostridium medium (RCM).” Bacillus and Clostridium are two different genera. Confused, please explain it and rewrite this sentence. The abstract section does not render the general significance and conceptual advance of the work clearly accessible to a broad readership, and the logic of the abstract is confused.

3. In Short title, “Characteristics of rabbit Clostridium sartagoforme”, please rewrite.

4. Please insert line numbers to ensure easy referencing during the reviewing process.

5. The Introduction should state the purpose of the investigation and give a short review of the pertinent literature, please include more details.

6. In Isolation and culture methods, “The beaker was placed in a 80 ℃ water bath for 10 minutes to kill the non-spore bacteria;” Is the water bath time too short?

7. Identification of C. Sartagoforme should include morphological identification, physiological and biochemical characterization, and molecular identification. Please clarify this issue and make appropriate adjustments to the content of the section.

8. In Heat resistance characteristics, “The survival rate was calculated as the ratio of other groups to the colony count of 100%. The calculation method of survival rate is unclear, please rewrite.

9. In “Gastric acid and bile salt tolerance”: 1) “and then the samples were inoculated in solid RCM and cultured anaerobically for 48 h before colony count”. This description is unclear. What’s the method? Plate count method? Please describe correctly. 2) The method of bile salt tolerance study is incorrect, please explain. The authors only determined the growth status of C. sartagoforme XN-T4 in different concentrations of bile salt solutions. It is not enough. Please add the survival rate of C. sartagoforme XN-T4 treated with different concentrations of bile salt solution for different time.

10. In Statistical analysis, what is the repeating unit of the experiment? What’s the expression of the results? The results were calculated only by SPSS? Please add the information.

11. In Isolation and identification of C. sartagoforme, the 16S rDNA gene sequence of the strain should be submitted to GenBank to obtain accession number. The 16S rDNA gene sequence and BLAST result of the strain can be deleted. When constructing the phylogenetic tree, what is the method? Please add confidence in the phylogenetic tree and make a clear figure.

12. Fig 5, the authors wrote “Characteristic of C. sartagoforme XN-T4 on Congo-CMC.” Is this 5 duplicates? Why are the sizes of the hydrolysis circles different? Please explain. It was strongly suggested that the authors listed the cellulose hydrolysis zone of C. sartagoforme.

13. In Growth characteristics, “this drop lasted for about 9 h” change to “this drop lasted for about 10 h”.

14. In Discussion, “Based on sugar alcohol fermentation experiments and gelatin liquefaction experiments for molecular identification, the isolated bacteria was further identified as C. sartagoforme (Shen and Chen., 2016).” Confused, please rewrite this sentence.

15. The citation of references is not standardized, please correct it.

16. In Discussion, “bile salt in bile accounts for 1% - 2%”. However, what the authors tested in the experiment was the growth of C. sartagoforme XN-T4 in 0.1% to 0.4% bile salt solution. It is recommended that the authors test the survival rate of C. sartagoforme XN-T4 after being treated in 1-2% bile salt solution for a certain period of time.

Reviewer #2: Thank you for the opportunity to review this manuscript. The authors describe the first, to the best of my knowledge, known isolation of Clostridium sartagoforme from the stool of a Hyla rabbit and suggest its potential utility as a microbial feed additive. Previously, Clostridium sartagoforme has been isolated from the groundwater, human stool samples, soil samples, and buffalo rumen. There is a lot of impressive work within the following document to identify the isolate as Clostridium sartagoforme, yet there is a lack of direction in the manuscript. What was the purpose of targeting Clostridium species in general? Why was this specific colony morphology selected? What motivated the authors to search for a microbial feed additive for rabbits? Additionally, the majority of the discussion section restates results but does little to contextualize this data towards the stated goal of developing a feed additive. While the data is thorough, the lack of explanation regarding the motivation of the study and minimal discussion limits the ability of the manuscript to effectively communicate with the reader.

Reviewer Comments:

1. Introduction

1.1. The initial setup of this paper relating to microbial feed additives was intriguing. However, following this introduction, there is little mention of this same notion. I will address this further down in the manuscript, but there is a lack of a major theme for this manuscript. What were the motivations for isolating Clostridium sartagoforme?

2. Materials and Methods

2.1. Overall, the methods seem appropriate.

2.2. Paragraph 5; line 2 -- It is stated: “the colonies that met the characteristics continued to be anaerobic cultured with RCM…” Could you please expand upon which characteristics you were looking for? I assume you were looking for common characteristics of Clostridium sartagoforme, but this has not been made clear. This may be further helped through further expansion of the introduction section as mentioned above.

2.3. Paragraph 7; line 4 -- For further reproducibility, please consider providing detail into the specific 2X mix used in the amplification of the 16S rRNA gene.

2.4. Table 1 -- Please revise the formatting of the PCR reaction conditions for readability

3. Results

3.1. Could the authors lend some insight into their naming conventions for each strain?

3.2. Table 6: How was the determination made between “++ good growth” and “+ growth”? Can the authors please clarify or provide a quantitative measure for how this determination was made?

4. Discussion

4.1. Paragraph 2; line 6 -- Up until this point, the authors have utilized numbered citations. However, the citation here is formatted differently. The journal requires numbered citations, please revise to match journal requirements.

4.2 Paragraph 7; line 6 – The authors state that the identification of the growth characteristics of C. sartagoforme XN-T4 “provide an important guarantee for the next step of scientific research and reasonable preparation of microbial agents for production tests”. The meaning behind this sentence is ambiguous and difficult to discern. Please consider revising for clarity.

4.3 Overall, there are many valuable threads of knowledge in the discussion section. However, there is a lack of a unifying flow of the data. In its current state, the discussion reads much like a rehashing of the results with the occasional relating to previous work with C. sartagoforme. While the authors state in the abstract that their study provides evidence of the potential utility of C. sartagoforme as a microbial food additive, there is essentially no discussion of the results that help support this point. Additionally, and even more critically, there is little discussion as to why C. sartagoforme would be beneficial as a microbial food additive.

**********

6. 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.

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Reviewer #1: No

Reviewer #2: No

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21 Mar 2021

Response to reviewers-3

1.Ren Zhanjun was the specific recipient of the five fundings [ (1) Shaanxi Province Science and Technology Project (K3310216062); (2) Shaanxi Province Agricultural Science and Technology Innovation and Research Project (16NY-108); (3) Shaanxi Province Agricultural Science and Technology Innovation Transformation Project (NYKJ-2020-YL-16); (4) Yangling Demonstration Zone Industry-University-Research Collaborative Innovation Major Project (1017cxy-15); (5) Key R&D project of Shaanxi Province (2018ZDXM-NY-041)] .

2.The methods of animal experiments were accordance with the relevant regulations by the Committee on Laboratory Animals of Northwest Agriculture & Forest University (NO. 0369/2018), and the pdf of animal experiments permission had been added as “supporting information”.

Submitted filename: Response to reviewers-3.doc

Click here for additional data file.

5 May 2021

PONE-D-20-28305R1

Isolation, identification and biological characteristics of Clostridium sartagoforme from rabbit

PLOS ONE

Dear Dr. Ren,

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 Jun 19 2021 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.

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We look forward to receiving your revised manuscript.

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Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

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 #1: All comments have been addressed

Reviewer #2: (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 #1: No

Reviewer #2: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: 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 #1: Yes

Reviewer #2: 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 #1: Yes

Reviewer #2: No

**********

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 #1: (No Response)

Reviewer #2: Thank you for the opportunity to once again review this manuscript. The authors have made a lot of improvements with this revision. The addition of the rabbit experiments is a fantastic addition to this manuscript and helps to move this manuscript beyond a potential use in a live animal model and probes at whether it is safe in trials involving rabbits. Despite the improvements, this manuscript is not yet ready for publication. One of the most glaring omissions is in the references provided. There are numerous instances throughout the manuscript that would benefit from the addition of some further literature review. Additionally, some further explanation and clarification are required before this manuscript will be ready for evaluation.

Abstract: It would benefit the paper if information concerning the rabbit testing of C. sartagoforme was added to the abstract

Line 50: Please consider adding a reference for this sentence.

Line 51: Please consider adding a reference for this statement.

Line 54: Please consider adding a reference for this statement concerning Bacillus.

Line 107: Please consider adding a reference for this statement. It may also be prudent to further explain this and the mechanism (over-decolorization) in some more detail.

Line 112: For the sake of reproducibility, could the authors please provide some further details on the genomic DNA extraction kit that was used?

Line 162: This difficult is difficult to read and should be rewritten (especially the end) for further clarity.

Line 172: It is unclear to me exactly how many rabbits were in each experimental group. Please consider revising this section for more clarity. A simple figure may also help this portion be more easily interpreted.

Line 193: Can you please describe the statistical tests that were used to compare the control and treatment groups for the trials including the rabbits?

Table 3: C. sartagoforme should have returned a negative result for the utilization of sorbitol. Were any steps taken to control for potential contamination in the experiments?

Line 282: As mentioned previously, how were significant differences between treatment groups of rabbits evaluated?

Table 7: It is curious that the diarrhea rate and mortality rate for group A are identical. Additionally, the standard error of the means appears to be very large for the diarrhea rate, this causes some concern for the conclusions that are drawn here. This concern may be alleviated once the analysis performed is described in more detail.

Line 294: It would be beneficial to add a citation for this sentence.

Line 303: The use of the term “molecular clock” is interesting here. I’m not sure that this is the best term to describe this. It seems that “molecular barcode” or “molecular identifier” would be some potential alternatives.

Line 314: It would be beneficial for a citation to be added here.

Lines 330-334: The manuscript would be improved if some references were added for each of these statements.

Line 334: There appears to be some typos and confusion in this sentence.

Line 341: It would be beneficial for a citation to be added here.

Line 346: It would be beneficial for a citation to be added here.

Line 355: It would be beneficial for a citation to be added here.

Line 371-374: Both of these references are for studies involving mice. Do these statements hold true for rabbits as well?

Line 385: This concluding sentence seems weirdly informal and would benefit from some rewriting.

**********

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 #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: Reviewers comments-2021,4,25.docx

Click here for additional data file.

16 Jul 2021

Response to reviewers-4

Abstract: It would benefit the paper if information concerning the rabbit testing of C. sartagoforme was added to the abstract

The sentence of “Moreover, animal testing indicated that this strain has no adverse effects on the morbidity and mortality of rabbits.” has been added.

Line 50: Please consider adding a reference for this sentence.

Citations has been added:

[1] Ayyat MS, Al-Sagheer AA, Abd El-Latif KM, Khalil BA. Organic Selenium, Probiotics, and Prebiotics Effects on Growth, Blood Biochemistry, and Carcass Traits of Growing Rabbits During Summer and Winter Seasons. Biol Trace Elem Res. 2018 Nov;186(1):162-173. doi: 10.1007/s12011-018-1293-2. Epub 2018 Mar 7. PMID: 29516355.

[2]Pogány Simonová M, Chrastinová Ľ, Lauková A. Autochtonous Strain Enterococcus faecium EF2019(CCM7420), Its Bacteriocin and Their Beneficial Effects in Broiler Rabbits-A Review. Animals (Basel). 2020 Jul 14;10(7):1188. doi: 10.3390/ani10071188. PMID: 32674281; PMCID: PMC7401553.

[3]Elghandour MMY, Tan ZL, Abu Hafsa SH, Adegbeye MJ, Greiner R, Ugbogu EA, Cedillo Monroy J, Salem AZM. Saccharomyces cerevisiae as a probiotic feed additive to non and pseudo-ruminant feeding: a review. J Appl Microbiol. 2020 Mar;128(3):658-674. doi: 10.1111/jam.14416. Epub 2019 Sep 8. PMID: 31429174.

[4]Zhou Y, Ni X, Wen B, Duan L, Sun H, Yang M, Zou F, Lin Y, Liu Q, Zeng Y, Fu X, Pan K, Jing B, Wang P, Zeng D. Appropriate dose of Lactobacillus buchneri supplement improves intestinal microbiota and prevents diarrhoea in weaning Rex rabbits. Benef Microbes. 2018 Apr 25;9(3):401-416. doi: 10.3920/BM2017.0055. Epub 2018 Jan 30. PMID: 29380642.

[5]Wang J, Ni X, Wen B, Zhou Y, Liu L, Zeng Y, Zhao W, Khalique A, Wang P, Pan K, Yu Z, Jing B, Liu H, Zeng D. Bacillus strains improve growth performance via enhancing digestive function and anti-disease ability in young and weaning rex rabbits. Appl Microbiol Biotechnol. 2020 May;104(10):4493-4504. doi: 10.1007/s00253-020-10536-9. Epub 2020 Mar 19. PMID: 32193576.

[6]El-Deep MH, Dawood MAO, Assar MH, Ahamad Paray B. Aspergillus awamori positively impacts the growth performance, nutrient digestibility, antioxidative activity and immune responses of growing rabbits. Vet Med Sci. 2021 Jan;7(1):226-235. doi: 10.1002/vms3.345. Epub 2020 Sep 9. PMID: 32902158; PMCID: PMC7840208.

Line 51: Please consider adding a reference for this statement.

Citations has been added:

[7]Wang Z. Preparation of Lactic acid bacteria Preparation an its Preliminary Application in Weaned Piglets [dissertation]. Harbin: Northeast Agricultural University; 2019.

[8]Bintsis T. Lactic acid bacteria as starter cultures: An update in their metabolism and genetics. AIMS Microbiol. 2018 Dec 11;4(4):665-684. doi: 10.3934/microbiol.2018.4.665. PMID: 31294241; PMCID: PMC6613329.

Line 54: Please consider adding a reference for this statement concerning Bacillus.

Citation has been added:

[9]Hu JH. Handbook of Medication. 4th ed. China Beijing: Jundun Press; 2009. Chinese.

Line 107: Please consider adding a reference for this statement. It may also be prudent to further explain this and the mechanism (over-decolorization) in some more detail.

Citation has been added:

[12]Shen P, Chen XD. Microbiology. 8th ed. Beijing: Higher Education Press; 2016. Chinese.

Explain: Clostridium is generally positive for Gram staining, but the cell wall structure of the old bacteria would become loose if the culture time is too long, and the result of Gram negative may appear after staining.

Line 112: For the sake of reproducibility, could the authors please provide some further details on the genomic DNA extraction kit that was used?

Details on the bacteria genomic DNA extraction kit that was added：Takara Bio Inc., Shiga, Japan.

Line 162: This difficult is difficult to read and should be rewritten (especially the end) for further clarity.

The methods were clarified as：

After incubation, bacteria were washed three times with phosphate-buffered saline (PBS; pH 7.2), 10-fold serial dilutions were made, and then, colony-forming units were counted on RCM agar plates after 48 h of incubation at 37 ℃. Tolerance was expressed as a percentage of the number of colony-forming units after incubation in artificial gastric acid relative to that of the normal RCM.

Line 172: It is unclear to me exactly how many rabbits were in each experimental group. Please consider revising this section for more clarity. A simple figure may also help this portion be more easily interpreted.

The methods were clarified as：

The experiment was conducted in a completely randomized design with 3 treatments, and every treatment with 3 replicates, 18 rabbits in each replicate (162 Rex rabbits 35-day-old ) that included the following treatments.

Line 193: Can you please describe the statistical tests that were used to compare the control and treatment groups for the trials including the rabbits?

Mortality and diarrhea rate of rabbits were analysised by one-way analysis of variance with SPSS 22.0 software (SPSS Inc., Chicago, IL, USA).

Table 3: C. sartagoforme should have returned a negative result for the utilization of sorbitol. Were any steps taken to control for potential contamination in the experiments?

The bacteria were inoculated in the clean bench, and the biochemical reaction tube was liquid sealed with sterilized liquid paraffin and covered with parafilm.

Line 282: As mentioned previously, how were significant differences between treatment groups of rabbits evaluated?

Data of mortality and diarrhea rate have been reanalyzed, and the significant differences between treatment groups of rabbits have been marked.

Table 7: It is curious that the diarrhea rate and mortality rate for group A are identical. Additionally, the standard error of the means appears to be very large for the diarrhea rate, this causes some concern for the conclusions that are drawn here. This concern may be alleviated once the analysis performed is described in more detail.

Data of mortality and diarrhea rate have been reanalyzed, but the standard error of the means still large, especially the numerical value of mortality are the same in different treament, the reason is that only 1 rabbit died in each group. Raw Data of Mortality and Diarrhea Rate as follow:

Raw Data of Mortality and Diarrhea Rate

Total number of rabbits Group Mortality Diarrhea rate

162 A Replicate 1 5.56 0.79

Replicate 2 0 0.79

Replicate 3 0 0.48

B Replicate 1 5.56 0.95

Replicate 2 0 0.79

Replicate 3 0 0.79

C Replicate 1 0 1.11

Replicate 2 5.56 0.63

Replicate 3 0 0.49

Notes: A, B and C respectively represent the amount of bacteria added according to the body weight of Rex rabbits: 108 CFU/Kg, 106 CFU/Kg and medium mixture.

Line 294: It would be beneficial to add a citation for this sentence.

Citation has been added:

[18]Sun XM. Effect of L-arginine and N-carbamylgluatmate on small intestinal villi development and cecal microbiota composition of young rabbits [dissertation]. Changchun: JiLin University; 2019.

Line 303: The use of the term “molecular clock” is interesting here. I’m not sure that this is the best term to describe this. It seems that “molecular barcode” or “molecular identifier” would be some potential alternatives.

Molecular clock had been changed as molecular identifier.

Line 314: It would be beneficial for a citation to be added here.

Citation has been added:

[20]Chen JD, Huang QY. Animal husbandry microbiology. 6th ed. China Beijing: Agriculture Press; 2017. Chinese.

Lines 330-334: The manuscript would be improved if some references were added for each of these statements.

Citations has been added:

Line 330:

[24]Ouyang WQ. Animal Physiology. Beijing: Science Press; 2021. Chinese.

Line 331:

[25]Guo ZQ, Wang B, Kuang LD, Yang R, Li CY, Zheng J. Gastrointestinal development and cecal cellulase activity in Qixing and New Zealand rabbits. Chinese Journal of Animal Science. 2021,57(03):130-133. Chinese

Line 332:

[26]Simonová MP, Chrastinová L, Kandricáková A, Kubašová I, Formelová Z, Chrenková M, Miltko R, Belzecki G, Strompfová V, Lauková A. Enterocin M and Sage Supplementation in Post-weaning Rabbits: Effects on Growth Performance, Caecal Microbiota, Fermentation and Enzymatic Activity. Probiotics Antimicrob Proteins. 2020 Jun;12(2):732-739. doi: 10.1007/s12602-019-09584-z. PMID: 31414382.

Line 334:

[27]Gu ZL, Qing YH, Ren KL. China rabbit Science. China Beijing: China Agriculture Press; 2013. Chinese.

Line 334: There appears to be some typos and confusion in this sentence.

This sentence has been changed as:

The transparent circle on the CMC-Congo Red plate indicates that the bacterium can decompose cellulose, it means that C. sartagoforme XN-T4 has the potential to improve the digestibility of cellulose for herbivores as a microbial feed additive.

Line 341: It would be beneficial for a citation to be added here.

Citation has been added:

[28]Dong XZ, Cai MY. System Identification Manual of Common Bacteria. Beijing: Science Press; 2001. Chinese.

Line 346: It would be beneficial for a citation to be added here.

Citation has been added:

[29]Yu LJ. Principles and Technology of Fermentation Engineering. Beijing: Higher Education Press; 2021. Chinese.

Line 355: It would be beneficial for a citation to be added here.

Citation has been added:

[29]Yu LJ. Principles and Technology of Fermentation Engineering. Beijing: Higher Education Press; 2021. Chinese.

Line 371-374: Both of these references are for studies involving mice. Do these statements hold true for rabbits as well?

These studies are mainly on mice, and there are no rabbits, so I have to refer to mice here.

Line 385: This concluding sentence seems weirdly informal and would benefit from some rewriting.

The conclusion rewrited as:

A strain of Clostridium sartagoforme XN-T4 was isolated from Hyla rabbit feces by RCM medium. Through colony characteristics and cell morphology, combined with physiological and biochemical methods and PCR identification. The strain has good stress resistance, can decompose cellulose, and has no obvious toxic effect on rabbits. It may be a candidate for microbial additives to improve the digestibility of cellulose in rabbits. Futher more, for its secondary metabolite composition, safety and in vitro and in vivo metabolic characteristics still need experimental research.

Reviewers' comments:

1.This manuscript reports the isolation, identification and biological characteristics of Clostridium sartagoforme from rabbit. However, it is not rigorous and unscientific. For using as microbial feed additive, the authors should add more research data on the probiotic effect and safety of Clostridium sartagoforme in vitro.

Dear reviewer, I would like to further the research on this strain. However, as I have graduated, I have no conditions and equipment to invest in this field. At present, I am applying for a doctor's degree, after getting the offer, I plan to continue the research on the probiotic effect and safety of Clostridium sartagoforme in vitro.

2. In Introduction, the authors propose that Clostridium sartagoforme had potential properties as a microbial food additives. However, following this introduction, there is little mention of this same notion. The authors should give a review of the pertinent literature, please include more details. What were the motivations and purpose for isolating Clostridium sartagoforme?

The motivations and purpose were to isolate a strain suitable for rabbits, which can resist the effect of digestive juice and improve the digestibility of cellulose in rabbits. By adding this strain, the economic benefit of rabbit breeding can be improved.

3. In Discussion, while the authors state in the abstract that their study provides evidence of the potential utility of Clostridium sartagoforme as a microbial food additive, there is essentially no discussion of the results that help support this point. Additionally, and even more critically, there is little discussion as to why Clostridium sartagoforme would be beneficial as a microbial food additive.

The reason for Clostridium sartagoforme would be beneficial as a microbial food additive as fellow:

The strain has no obvious toxic effect on rabbits, to a certain extent, it indicates that the bacterium is not a pathogen.

The strain has good stress resistance which can resist the effect of digestive juice, this means that the bacteria may reach the rabbit's cecum to play a role.

The strain can decompose cellulose, it is possible that the strain can improve feed utilization by decomposing cellulose.

4. All taxa names (species names, genus names, and names of higher categories) should be italicized.

Taxa names had been italicized in the article.

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

23 Aug 2021

PONE-D-20-28305R2

Isolation, identification and biological characteristics of Clostridium sartagoforme from rabbit

PLOS ONE

Dear Dr. Ren,

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 Oct 07 2021 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'.

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Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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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 #1: Partly

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: (No Response)

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Submitted filename: Reviewers comments-2021,7,27.docx

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7 Oct 2021

Response to Reviewer#5:

Point 1: In the last review, I commented “the authors should add more research data on the probiotic effect and safety of Clostridium sartagoforme in vitro”. The author did not realize the importance of its safety and effectiveness. In any case, as microbial a feed additive, the safety of probiotics is very important. If there is no guarantee that Clostridium sartagoforme is safe, all subsequent studies will be futile. Therefore, some basic safety tests such as antibiotic susceptibility test, hemolytic test, and toxicity test are necessary.

Response 1: Thank you for your instructive suggestions. The studies of diarrhea rate and mortality rate on rabbits were used to demonstrate the safety of this strain, and further experiments such as cellulase production, components of secondary metabolites, antibiotic susceptibility test, hemolytic test, toxicity test, blood biochemical inedxes and organ coefficients of experimental animals will be carry out.

Point 2: In the last review, I commented “The authors should give a review of the pertinent literature, please include more details.” in the introduction. The author did not make changes in accordance with the comments, and the explanation given was unreasonable. The introduction should provide with sufficient background information to introduce the reader into the reported study. If the author is not clear about the relevant research progress of Clostridium sartagoforme, how does the author think that it can be used as a microbial food additive? A review of the pertinent literature is necessary and it can well reflect the authors' motivations and purpose for isolating Clostridium sartagoforme.

Response 2: Thank you for your careful reading of our manuscript. The introduction has been rewrote as follow:

Rabbits are herbivores, there are abundant cellulose degrading bacteria resources in rabbit intestinal tract, because they rely on the microorganisms in the caecum to digest cellulose but not produce cellulase by themselves. In addition, cellulose degrading bacteria isolated from animals have natural advantages in the utilization of crude fiber feed. Most of them are inherent bacteria in the intestinal tract, which are easier to adapt to the intestinal environment of the animal and play a role.

Clostridium sartagoforme is an anaerobic spore producing bacterium, which determines that the bacterium has a natural advantage in resisting the effects of digestive juice and adapting to the anaerobic environment of the digestive tract. Researches have shown that the strain can decompose microcrystalline cellulose, carboxymethyl cellulose and chitin, and its metabolites include formic acid, acetic acid, propionic acid, lactic acid, butyric acid, isovaleric acid and hydrogen. Simůnek J et al. isolated C. sartagoforme from feces of horse, and this strain has high exocellular activity of N-Acetylglucosaminidase. Zhang JN isolated Clostridium sartagoforme FZ11 from cow dung compost, using urea as a nitrogen source and raw corn stalk as a substrate, confirming that this strain could decompose cellulose and have good hydrogen production characteristics. In Nathani Neelam M and coleague’s research, Clostridium sartagoforme AAU1 was isolated from Surti buffalo rumen liquor, and they pointed out that VFAs produced by the bacteria play an important role in animal health maintenance through genomic analysis. At present, the research about C. sartagoforme isolated from digestive tract or feces of rabbits has not been reported, and there is little information on the biological characteristics of this strain. In this experiment, C. sartagoforme was isolated and identified from rabbit feces, and its physiological, biochemical and stress resistance characteristics were preliminarily studied, which help to provide reference for the further development of rabbit microbial additives.

Point 3-(1): Whether there are other more reasonable explanations for changing from Gram-positive to Gram-negative of Clostridium sartagoforme.

Response 3-(1): Thank you for your careful reading of our manuscript. For this phenomenon, only this point of view is currently being seen.

Point 3-(2): In Line 162, reviewers-4 commented “This difficult is difficult to read and should be rewritten (especially the end) for further clarity”, the authors clarified the methods, but there are still some details that you need to add. After 10-fold serial dilutions, how many dilutions should be taken, whether it is spread or spotted, and how is the colony-forming units calculated?

Response 3-(2): I'm sorry for the article writing problem. Samples were diluted up to 10-8, 50 μL of the diluted cultures was spread on RCM agar plates and incubated for 48 h at 37 ℃, 6 replicates for each sample, followed by colony counting. The survival rate (%) in gastric acid was calculated with the mean of colony counts of the plates after treatment with respect to their mean before treatment. The results were presented in mean number of surviving bacteria ± standard deviation.

Point 3-(3): In Line 172, reviewers-4 commented “It is unclear to me exactly how many rabbits were in each experimental group.”, but there are still some details that you need to add. Are female rabbits or male rabbits used in the experiment, and what is the ratio?

Response 3-(3): I apologize for the ambiguity in the description. A total of 162 35-day-old Rex rabbits (half males and half females) were randomly divided into 3 groups with 9 replicates per group and 6 rabbits per replicate.

Point 3-(4): The results of diarrhea rate and mortality are quite different before and after, please confirm the accuracy and authenticity of the data. The formula for calculating the diarrhea rate may be wrong, please confirm.

Response 3-(4): Thank you for your valuable advice. The diarrhea rate has been checked to be correct, but the formula for diarrhea rate is indeed not accurate enough, it has been changed as:

Point 3-(5): In Line 371-374, reviewers-4 commented “Both of these references are for studies involving mice. Do these statements hold true for rabbits as well?”, the authors responded “These studies are mainly on mice”. Please change references for studies related to rabbits.

Response 3-(5): Thank you for your careful work. The reference about “gastric juice and pH in stomach” had been changed to those researches on rabbits. The results of this study are indeed very different in rabbits and mice. I am ashamed of the previous erroneous quotation of references and sincerely thank you for your suggestions.

Point 3-(6): Reviewers-4 suggested that the author added a lot of references, but most of the newly added references are in Chinese. Please replace some of the references with English references.

Response 3-(6): According to the reviewer’s comments, most of the references in Chinese had been changed to English references.

Submitted filename: Response to Reviewer #5.docx

Click here for additional data file.

26 Oct 2021

Isolation, identification and biological characteristics of Clostridium sartagoforme from rabbit

PONE-D-20-28305R3

Dear Dr. Ren,

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,

Alistair K Brown

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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 #1: All comments have been addressed

**********

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 #1: Yes

**********

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

Reviewer #1: Yes

**********

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 #1: 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 #1: 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 #1: (No Response)

**********

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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 #1: No

Submitted filename: Reviewers comments-2021,10,25.docx

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5 Nov 2021

PONE-D-20-28305R3

Isolation, identification, and biological characteristics of Clostridium sartagoforme from rabbit

Dear Dr. Ren:

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.

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PLOS ONE