Antibiotic cocktail followed by Clostridium butyricum (CBM588) supplementation improves colonic anastomotic healing in mice

doi:10.21203/rs.3.rs-2448867/v1

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Antibiotic cocktail followed by Clostridium butyricum (CBM588) supplementation improves colonic anastomotic healing in mice

https://doi.org/10.21203/rs.3.rs-2448867/v1

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Background

Microbiota manipulation through preoperative probiotic administration with Clostridium butyricum (CBM588) has shown promising results in reducing surgical site infections, but the resulting changes in gut microbiota are unclear. This study, using 16S ribosomal RNA metagenomic analysis, aimed to observe microbiome changes after CBM588 administration before murine colonic anastomosis.

Materials and methods

Eighteen C57BL/6 mice were divided into three groups. All mice, including group A as the control group, received oral neomycin and metronidazole before surgery. Group B received six consecutive days of CBM588 before surgery. Group C received kanamycin, gentamycin, colistin, metronidazole, and vancomycin, followed by six consecutive days of CBM588 before surgery.

Results

Compared with groups A and B, group C had significantly lower adhesion scores (p < 0.001), better postoperative performance (p < 0.05) and anastomotic healing scores (p < 0.001), and lower anastomotic necrosis areas (p < 0.05). Metagenomic analysis revealed that group C was enriched with the order Clostridiales, family Clostridiaceae, and genus Clostridium, with low bacterial diversity and composition.

Conclusions

Using a murine colonic anastomosis model, a preoperative antibiotic cocktail followed by CBM588 supplementation effectively decreased bacterial diversity and increased the percentage of Clostridiaceae. Associated outcomes included lower intra-abdominal adhesion scores, better postoperative performance and anastomotic healing scores, and lower anastomotic necrosis areas.

Clostridium butyricum

bowel preparation

anastomotic healing

16S rRNA analysis

antibiotic cocktail

Postoperative anastomotic leak (AL), the most severe form of surgical site infection (SSI) after colorectal surgery, is associated with high morbidity and mortality^1,2. According to a nationwide cohort study in the United States, 11.06% of SSIs were identified following colorectal resection, of which 33.3%−56.1% may be attributed to organ-space SSIs in patients with cancer³, which is a well-established proxy for AL⁴. Since SSI is a costly burden to both healthcare providers and patients, SSI prevention bundles have been developed to lower morbidity and mortality and to improve surgical quality in colorectal surgery^5,6. In a recent meta-analysis of SSI bundles in colorectal surgery, mechanical bowel preparation combined with oral antibiotics was demonstrated to be highly correlated with organ-space SSI reduction⁷. However, a growing threat to the effectiveness of traditional preoperative antibiotic use before colorectal surgery has emerged in recent years⁸.

In the last two decades, the inclusion of probiotics during the perioperative period has been demonstrated to be beneficial in reducing infectious complications in elective intra-abdominal surgery⁹, resulting in a better balance of beneficial-to-pathogenic microorganisms after colorectal surgery¹⁰. In eastern Asia, Clostridium butyricum (CBM588) is a widely used and commercially available probiotic for the treatment of antibiotic-associated diarrhea¹¹. However, it has never been used as a probiotic backbone in the bowel preparation regimen for colorectal surgery. CBM588 secretes substances to suppress the growth of extended-spectrum β-lactamase-producing E. coli, reduces β-lactamase enzymatic activity and gene expression, and inhibits the transmissibility of antibiotic resistance genes in vitro¹². In addition, cell-free supernatants derived from CBM588 exert antimicrobial and antibiofilm effects on A. baumannii¹¹. Recently, CBM588 was proposed as an antimicrobial resistance countermeasure¹³. Adding CBM588 to the bowel preparation regimen to inhibit the colonization of multidrug-resistant bacteria in the human gut might be an attractive approach to decrease the occurrence of AL and SSI after colorectal surgery.

The manipulation of the gut microbiota (GM) through the addition of preoperative oral antibiotics and probiotics to reduce infectious complications in colorectal surgery may be the key to clinical success in AL and SSI prevention¹⁴. In mouse models, reproducible GM changes through different regimens of antibiotic cocktails have been frequently used^15,16 to deplete normal GM and mimic the sterile gut of germ-free mice, to explore the effects of GM in pathological conditions^17–24, such as Clostridium difficile infection^18,19, ulcerative colitis²³, and colon tumorigenesis²⁴.

The aims of this study were to manipulate GMs in different clinical scenarios before colon anastomosis surgery, which include 1) preoperative oral neomycin and metronidazole, 2) CBM588 inoculation followed by preoperative oral neomycin and metronidazole, and 3) antibiotic cocktail followed by CBM588 inoculation, followed by preoperative oral neomycin and metronidazole for colonic anastomosis. The consequence and corresponding effects on anastomotic healing were observed in an established murine model²⁵. Colonic anastomotic healing was evaluated by histological sectioning and hematoxylin and eosin (H&E) staining. GM changes were evaluated by 16S ribosomal RNA (rRNA) amplicon and shallow metagenomic sequencing (SMS) analysis. These results may provide an understanding of the ideal bowel preparation regimen before murine colonic anastomosis surgery.

Eighteen mice were divided into groups A, B, and C. A summarized comparison among groups A, B, and C is presented in Table 1. Detailed explanations of the clinical parameters are provided below.

Mice Survival Rate And Body Weight Change

No mortality was observed in group C (Fig. 1). In group A, one mouse exhibited a drastic decrease in body weight and died on the 6th day after surgery. In group B, one mouse also showed a drastic decrease in body weight before dying on the 4th day after surgery. Intra-abdominal abscesses were identified when the deceased mice were sacrificed. However, the general body weight change and mortality rates did not achieve statistical significance among the three groups.

Adhesion Scores

Significantly fewer intra-abdominal adhesions were noted in group C than in the other two groups (Table 1, compared to group A, p < 0.001; compared to group B, p = 0.006).

Postoperative Performance Scores

We modified a previously published scoring protocol, as listed in Supplementary Table 1. Based on the scoring data, we observed that group C had significantly better postoperative performances than group A (Fig. 2, p = 0.049).

Impact Of Different Treatments On The Anastomotic Healing Score

Colonic anastomotic sections were harvested after the mice were sacrificed. The anastomotic healing score (AHS) of the colonic anastomoses among the three groups were compared using histological sections and H&E staining (Fig. 3A). As shown in Fig. 3B, the AHS was significantly higher in the colonic anastomosis of group C than in that of group A (p < 0.01) or group B (p = 0.003). In group A, although the colonic anastomosis section contained fibroblasts and some restored serosa, submucosal formation was not commonly observed. In contrast, group C showed extensive (83%) mucosal and submucosal healing.

Since organ-space SSI, is associated with local tissue damage, microorganism infection, and acute inflammation, causing local necrosis and impeding wound healing, we also scored the percentage of necrotic areas over the colonic anastomosis sites under H&E staining for all three groups. The results are presented in Fig. 3C. Extensive inflammatory cells in the tissue where cells had lost their structural integrity and morphology were observed in group A. In contrast, group C had significantly smaller necrotic areas than group A (p < 0.05). Based on the histological evidence, we suggest that the preoperative treatment in group C promoted the wound-healing process and reduced the SSI rate in the murine colonic anastomosis model.

Impact Of Different Treatments On The Gut Microbiome

The results of our 16S rRNA amplicon with SMS analysis showed the impact of different treatments on murine GM. First, there was no obvious difference in bacterial α-diversity between groups A and B. However, there was a significant decrease in α-diversity in group C compared to that in groups A and B (Fig. 4A). Second, the principal components analysis of β-diversity showed that the bacterial diversity of groups A and B were similar, but clearly distinct from that of group C (Fig. 4B). Compared with groups A and B, group C not only exhibited decreased bacterial diversity but also a significant difference in the composition of bacterial communities. Third, a significant change in murine GM after preoperative treatment in group C was noted compared to that in groups A and B (Fig. 5A). The relative abundances of the top 30 most prevalent bacterial families in mouse fecal samples are shown as bar graphs. A total of 477 amplicon sequence variants (ASVs) with a total frequency of 1197468 were obtained using the DADA2 (Divisive Amplicon Denoising Algorithm 2) pipeline. The intestinal microbial communities in each group consisted mainly of bacteria in the phyla Firmicutes (class Bacilli and Clostridia) and Bacteroidetes (class Bacteroidia), and 111 bacterial taxa were identified at the genus and species levels. Fourth, the differential abundance analysis showed that (1) group A was enriched with class Bacilli, order Lactobacillales, and family Lactobacillaceae; (2) group B was enriched with family Ruminococcaceae, genus Ruminococcus, and Clostirdia UCG-014; and (3) group C was enriched with orders Clostridiales, family Clostridiaceae, and genus Clostridium (Fig. 5B). In short, the administration of CBM588 in group B increased the abundance of the family Ruminococcaceae, genus Ruminococcus, and ClostirdiaUCG-014, compared to that in group A. Preoperative administration in group C radically changed the microbiota diversity and greatly enhanced Clostridiaceae abundance in the mouse gut, compared to that in the other two groups.

This study demonstrated that in a murine model, ingesting only a normal dosage of CBM 588 before colonic anastomosis surgery did not have beneficial effects on the AHS or resulted in a significant change in the GM in mouse feces. However, when an antibiotic cocktail followed by CBM588 ingestion was implemented preoperatively, compared with other groups, a dramatic change in GM with an improvement of intra-abdominal adhesion scores, postoperative performance, lower anastomotic necrosis areas, and better AHS were noted. Our results suggest an association between the preoperative manipulation of GM and improved colonic anastomotic healing in a murine model.

Since the introduction of the revolutionary method of bacterial characterization by Pace et al.²⁶, 16S rRNA-based characterization of bacterial species has been universally accepted as an accurate method for bacterial identification²⁷. 16S rRNA metagenomic analysis has provided an objective tool to explore bacterial diversity and composition under a wide variety of clinical scenarios, in which 16S rRNA amplicon with SMS analysis was widely adopted for economic affordability and rapid microbial detection strategy²⁸.To date, the use of 16S ribosomal RNA (rRNA) metagenomic analysis to study GM changes in the clinical setting have demonstrated the capability to analyze associations between GM changes and colorectal diseases²⁸, COVID-19 infections²⁹, prosthetic joint infections³⁰, chronic wound infections after wisdom tooth extraction³¹, detection of postoperative infection sources³², and the potential to influence SSI outcomes in colorectal surgery¹⁴. In this study, the 16S rRNA amplicon with SMS analysis was utilized to verify gut GM changes in mice. The consistency between the results of the 16S rRNA amplicon with SMS analysis and better colonic anastomosis healing suggests the applicability of its use in the clinical setting.

As presented in our study, antibiotic cocktail administration followed by CBM 588 ingestion resulted in significant changes in the fecal microbiome, including a significant decrease in α-diversity and a lower ASV number. In addition, β-diversity analysis showed a significant decrease in richness and composition compared with the control and CBM588 monotherapy groups. The family Clostridiaceae, which mainly colonizes the large intestine and affects bowel mucosa metabolism through its butyrate-producing ability^33,34, achieved a 15% relative abundance in the antibiotic cocktail group compared to 0.06% in the control group and 1% in the CBM 588 group. This major shift in GM may be associated with the beneficial effect of suppressing intestinal inflammation and providing antioxidant protection, as reported in previous studies^35,36. In other words, the lone addition of CBM588, using the same proportional dosage as that of a 60 kg man, into the preoperative setting of a murine colonic anastomosis would not have any beneficial effects in improving AHS. The respective 16S rRNA amplicon with SMS analysis demonstrated that the family Lachnospiraceae was abundant in the control group, and the family Ruminococcaceae and genus Ruminococcus were abundant in the CBM588 group, which was associated with anastomotic leaks³⁷. However, when an antibiotic cocktail was first administered to eradicate normal colonizing flora in mouse guts, the subsequently added CBM588, also using the same proportional dosage as that of a 60 kg man, clearly had more room to grow and become one of the dominant families in the bacterial composition of mouse feces. Therefore, we considered that the reduced intra-abdominal adhesion score, better postoperative performance score, and better AHS after murine colonic anastomosis surgery were closely related to the beneficial effects of abundant CBM588 in the mouse gut, and these results could be supported by the findings of the 16S rRNA amplicon with SMS analysis. Other supporting studies on the beneficial effects of CBM588 included the modulatory effect of neutrophils and cytokines through butyrate production³⁸ and its ability to protect the intestinal barrier in a microbiome dysbiosis model³⁹. The butyrate-producing characteristic of CBM588 has been demonstrated to have the potential to inhibit pathogenic bacteria^11,13,40. Butyrate was also demonstrated to improve anastomotic healing^41–43 in murine models. However, further studies are required to delineate the exact mechanisms behind our findings.

Studying the etiology of colonic anastomotic leaks through the lens of microbiology is a new and promising field⁴⁴, especially when the concept of probiotic addition in colorectal surgery has only emerged in recent decades¹⁰. Probiotics have been reported to have the potential to reduce SSIs and at the same time improve the time to first bowel movement and reduce the occurrence of abdominal cramps and diarrhea. However, these studies did not provide supporting evidence from the study of the microbiome in the human gut. The findings of our study highlight an important point: to achieve significant AHS and reduce subsequent SSI and mortality, preoperative bacterial diversity and composition in the gut should be changed from that in the control group. There are only two ways to achieve this goal, either probiotics need to be administered to mice at a much higher dosage, or as we did in this study, the normal gut flora have to be first eradicated using an antibiotic cocktail to make room for the subsequently added normal dosage of probiotics to grow and make a significant change in the bacterial composition of mouse feces. Therefore, the future implication of this study is to determine which method is more effective for GM manipulation to achieve better AHS in a murine colonic anastomosis model. We plan to prescribe a much higher dosage of preoperative probiotic regimens for mice, or we will routinely prescribe antibiotic cocktails followed by a regular dosage of preoperative probiotic regimens to compare AHS. Subsequently, we will also perform 16S rRNA metagenomic studies on the mouse feces for objective GM analysis.

This study had several limitations. First, the study was performed using a murine model. Compared with a real-world patient cohort, which will involve multiple confounding factors, a murine model has the advantages of unified sample characteristics, unified drug manipulation processes, and unified surgical procedures. However, the favorable results of this murine model cannot be directly applied to human cohorts. Second, this study used Clostridium butyricum as the probiotic species. In the real world, there exist many other probiotic species, including Bifidobacterium longum and Lactobacillus acidophilus, which are also proven to have the ability to improve anastomosis healing. Our favorable results cannot be generalized to other studies, and additional probiotic species should be explored in the future.

We demonstrated that, in a murine colonic anastomosis model, a preoperative antibiotic cocktail followed by CBM588 supplementation could effectively decrease the bacterial diversity of the feces and increase the percentage of Clostridiaceae, which was shown in a 16S rRNA amplicon with SMS analysis. The associated results included a lower intra-abdominal adhesion score, better postoperative performance score, lower anastomotic necrosis area, and better AHS, compared to standard preoperative oral neomycin and metronidazole bowel preparation. Our results suggest an association between manipulating the GM using a preoperative antibiotic cocktail followed by CBM 588 and improved colonic anastomotic healing in a murine model.

Preparation of the animals

We used 10 to 12-week-old, adult female C57BL/6 mice with an average weight of 20–25 g. A standard diet and water were provided for at least one week prior to all experiments to allow the mice to adapt to the local conditions and eliminate shipping stress. C57BL/6 mice were purchased from the National Cheng Kung University Animal Center, housed under standard laboratory conditions with a 12-h light-dark cycle, and provided food and water in a temperature-controlled room (22–24°C). All experiments were approved and reviewed by the National Cheng Kung University Animal Center Institutional Animal Care and Use Committee (IACUC-110157) and performed in accordance with relevant guidelines and regulations. The study is reported in accordance with ARRIVE guidelines.

Preoperative Antibiotic And Probiotic Bowel Preparations

Eighteen mice were divided into three groups, with six mice in each group, as shown in Fig. 6.

Group A was given a normal diet and water until the administration of the antibiotic bowel preparation (neomycin 0.38 mg/mouse and metronidazole 0.42 mg/mouse), which was based on the proportional calculation of dosage using a 60 kg man. Oral antibiotics were given one day before surgery. Group B was administered probiotics (Clostridium butyricum CBM588 0.05 mg/mouse/day, also based on a proportional calculation of dosage using a 60 kg man) via an intragastric tube once daily for 6 days before the antibiotic bowel preparation. Group C was initially given an antibiotic cocktail in water (kanamycin 0.4 mg/mL, gentamycin 0.035 mg/mL, colistin 0.057 mg/mL, metronidazole 0.215 mg/mL, and vancomycin 0.045 mg/mL) for 6 days to eradicate normal colonizing gut bacteria, which was frequently used in a standard murine model for Clostridum difficile infection research⁴⁵. After a 1-day break, group C was then given the probiotic CBM588 daily (Clostridium butyricum CBM588 0.05 mg/mouse/day, based on a proportional calculation of dosage using a 60 kg man) for 6 days before antibiotic bowel preparation. All fecal pellets were collected before antibiotic bowel preparation for 16S rRNA metagenomic analyses.

Creation Of Colorectal Anastomoses

All the surgeries were performed by a single surgeon (Po-Chuan Chen), who followed the standard operative procedures described by McKarthy et al²⁵. After general anesthesia was established with a single-dose intraperitoneal injection (50 mg/kg zoletil and 2.5 mg/kg xylazine), mice were prepared using the methods described previously²⁵. Through a midline incision down to the peritoneal cavity, the bowels were carefully moved to the left side to expose the descending and sigmoid colons. Transverse colotomy of the sigmoid colon was performed using sharp micro-scissors (Supplementary Fig. 1A). Four to five interrupted stitches of 7 − 0 nylon stitches were used to repair the colotomy (Supplementary Fig. 1B). To test the integrity of the anastomosis, we filled the abdomen with sterile saline and then slowly inflated the anastomosis with 1 cm³ of air by advancing a lubricated 18 G angiocatheter into the rectum. 3 − 0 nylon sutures were used to close the abdominal incision and the skin. The warmer was turned on during the entire procedure to ensure that the animals were warm and prevent the occurrence of postoperative hypothermia.

Postoperative Performance Evaluation

The mice were observed daily and scored for seven consecutive days after surgery. Score measurements were performed as described previously⁴⁶. In brief, the score points included weight change, behavior, posture, and pain (Supplementary Table 1).

Anastomotic Tissue Sample Harvesting

On day 7, the mice were sacrificed by placing them in a carbon dioxide gas box. Subsequently, the original abdominal incision was re-opened to explore the intra-abdominal contents. After carefully removing possible bowel-to-bowel adhesions, the segments above and below the bowel anastomosis were resected. Tissues were fixed in neutral buffered formalin for histological analysis and H&E staining.

Adhesion Score

Intra-abdominal adhesions were scored using a scoring system described previously⁴⁶. Examples of intra-abdominal adhesions are shown in Supplementary Fig. 2.

Anastomotic Healing Score

Histological changes were evaluated based on the AHS as described previously⁴⁶. Supplementary Table 2 provides details regarding the allocation of score points, including blood vessel ingrowth, fibroblasts, collagen formation, inflammatory cells, and healed layer description. Histological results were scored by a pathologist (Chien-Chin Chen) who was blinded to the treatment of the mice. Supplementary Fig. 3 shows the histological scoring of the anastomosis site.

Dna Extraction And Polymerase Chain Reaction

DNA was extracted from mouse feces, which were collected before antibiotic bowel preparation, using a fecal DNA extraction kit. ND-10000 (NanoDrop, Thermo Fisher Scientific, Waltham, MA, USA) was used to quantify the concentration of the purified DNA. Total DNA was stored at − 20 ºC in a refrigerator. For amplification of 16S ribosomal RNA gene variable regions 3 and 4, the forward primer (5’-ACT CCT ACG GGA GGC AGC AG-3’) and reverse primer (5’-ATT ACC GCG GCT GCT GG-3’) were used. PCR was conducted as described previously (5 min at 94°C, followed by 35 cycles of 30 s at 94°C, 1 min at 58°C, 30 s at 72°C, and 7 min at 72°C)⁴⁷. The amplification products were separated by 1.5% agarose gel electrophoresis, stained with DNA VIEW (BIOTOOLS CO., Ltd. New Taipei City, Republic of China), and quantified using a SmartView Pro 1200 Imager System (Major Science, Saratoga, CA, USA).

V3-v4 16s Metagenomic Analysis

A total of nine samples (Group con, pre, pro with triplicates) were used for 16S rDNA Amplicon Sequencing using Illumina Sequencing-by-Synthesis technology to produce 2 × 300 bp paired-end reads. The V3-V4 hypervariable region of 16S rDNA was amplified using bacterial-specific forward (5’-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG-3’) and reverse (5’-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3’) primer sets using the 16S Metagenomic Sequencing Library Preparation Kit (Illumina, San Diego, CA, USA). Indexed adapters were added to the amplicons using the Nextera XT Index Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Amplified DNA sizing accuracy was checked using the 4200 TapeStation System (Agilent Technologies, Santa Clara, CA, USA). After library construction, samples were mixed with the MiSeq Reagent Kit v3 (600-cycle) and loaded onto a MiSeq cartridge, and a 2 × 300 bp paired-end sequencing run was performed using the MiSeq platform (Illumina, San Diego, CA, USA). Sample preparation, library construction, and sequencing were performed by Welgene Biotech Co., Ltd. (Taipei, Republic of China).

Statistical analysis

Alpha diversity indices were compared using pairwise Kruskal–Wallis tests, in which the p value was adjusted using the Benjamini–Hochberg procedure. For β-diversity, pairwise analysis of similarities and permutational multivariate analyses of variance with 999 permutations were conducted and evaluated using principal coordinate analyses based on different distance matrices, where p-values were reported after Benjamini–Hochberg multiple testing correction.

Table 1

Comparison of clinical parameters among the three groups
	Group A^@	Group B^@	Group C^@	p value*
Survival rate (%)	83.3	66.7	100	0.317
Body weight change (%)	11.32 (4.58)	9.18 (3.85)	6.93 (2.50)	0.378
Adhesion score	7.4 (0.24)	6.75 (0.25)	5.375 (0.30)	< 0.001
Postoperative performance score	4 (1.79)	3.2 (2)	0	0.049
Anastomotic healing score (AHS)	9.4 (1.29)	13.25 (0.33)	16.88 (0.58)	< 0.001
^@Group A: control; Group B: CBM588; Group C: Antibiotic cocktail + CBM588
*Comparison between groups A and C
^#Data are presented as median (interquartile range, IQR)

Acknowledgement

We would like to thank Yi-Chen Tang for helping us prepare the operation room and the logistics associated with the operation.

Data availability statement: The datasets generated and analyzed during the current study are available in the GenBank repository, with ACCESSION NUMBER of OQ286544 - OQ287042. We provide a temporary link to our data submitted in the GenBank repository: https://drive.google.com/drive/folders/1fuugvzl4U3Dg9_nuY4yx_fCtyVBnAP3z?usp=share_link.

Author contributions: Chun-Hsien Chen and Jenn-Wei Chen contributed to data analysis, interpretation, and manuscript drafting. Chao-Han Lai contributed to critical manuscript revision. Chih-Ming Tsai contributed to data acquisition and uploading. Chien-Chin Chen contributed to data analysis and interpretation. Wei-Chen Lin and Po-Chuan Chen contributed to study conceptualization, data acquisition, animal experimentation, analysis and interpretation, manuscript drafting and final approval.

Additional information:

Conflict of interest statement: All authors declare no conflict of interest.

Funding supports: This study was supported by research grants from the National Cheng Kung University Hospital (NCKUH-11104067). The research was also supported in part by Higher Education Sprout Project, Ministry of Education to the Headquarters of University Advancement at National Cheng Kung University (NCKU). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. There are no competing interests.

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No competing interests reported.

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Antibiotic cocktail followed by Clostridium butyricum (CBM588) supplementation improves colonic anastomotic healing in mice

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Abstract

Background

Materials and methods

Results

Conclusions

Figures

Introduction

Results

Mice Survival Rate And Body Weight Change

Adhesion Scores

Postoperative Performance Scores

Impact Of Different Treatments On The Anastomotic Healing Score

Impact Of Different Treatments On The Gut Microbiome

Discussion

Conclusions

Methods

Preparation of the animals

Preoperative Antibiotic And Probiotic Bowel Preparations

Creation Of Colorectal Anastomoses

Postoperative Performance Evaluation

Anastomotic Tissue Sample Harvesting

Adhesion Score

Anastomotic Healing Score

Dna Extraction And Polymerase Chain Reaction

V3-v4 16s Metagenomic Analysis

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

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