Metagenomics reveals functional profiles of gut microbiota during the recovery phase of acute pancreatitis

Metagenomics reveals functional profiles of gut microbiota during the recovery phase of acute pancreatitis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Metagenomics reveals functional profiles of gut microbiota during the recovery phase of acute pancreatitis Xiao Song, Jia-Ning Li, Duan Wang, Zi-Ying Han, Xia-Xiao Yan, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5453055/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Gut microbiota play a critical pathogenic role in acute pancreatitis (AP). This study aimed to investiage the gut microbiota composition and function during the recovery phase of AP. Rectal swab samples obtained from 12 AP patients of varying severity in the acute and recovery phases were sequenced using shotgun metagenomic sequencing. The α-diversity, principal components, typing, and dominant microbiome composition were analyzed, followed by a difference analysis of gut microbiota composition and functional enrichment. In the recovery phase of AP, the microbial diversity remained decreased, and there were minimal difference in the structural diversity of the microbiome. There was an increasing tendency of beneficial bacteria (Bacteroidales) and a decreasing tendency of harmful bacteria (Enterococcus and Firmicutes) in the recovery phase of mild AP (MAP). However, in the recovery phase of moderately severe AP (MSAP) and severe AP, Enterococcus abundance increased compared with that in the acute phase. Some signaling pathways changed in the opposite direction in the recovery phase of MAP and MSAP compared to the acute phase. These results suggested that gut microbiome composition and function are associated with AP recovery, which may be used to develop strategies for the treatment and prognosis of AP. Biological sciences/Microbiology/Communities Health sciences/Gastroenterology/Gastrointestinal diseases/Pancreatic disease/Pancreatitis acute pancreatitis recovery phase shotgun metagenomics gut microbiota microbiota composition dysbiosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Acute pancreatitis (AP) is a common life-threatening disease of the pancreas. Non-mild AP has an acute onset, rapid progression, and poor prognosis [ 1 , 2 ]. The course of AP often lasts from tens of days to months, depending on disease severity. Based on its clinical characteristics, AP can generally be divided into three phases: acute reaction, progression, and recovery. While not all patients experience a three-phase course, every surviving patient undergoes a recovery phase [ 3 ]. Pancreatic regeneration and functional repair may occur during the acute reaction period. When inflammation occurs, organ repair and regeneration mechanisms are activated, which continue throughout the course of the disease. During the acute reaction and progression phases, the pathophysiological processes are the injury and necrosis of pancreatic cells as well as inflammatory responses. After entering the recovery period, pancreatic regeneration and functional repair become the primary pathophysiological processes with inflammatory responses gradually weakening or attaining a balance [ 4 , 5 ]. At this stage, relief of discomfort, regression of inflammation, and recovery of gastrointestinal function occur as responses to treatment and enhance the quality of life of patients [ 6 ]. However, a risk of deterioration exists. The human intestinal barrier system consists of the gut flora, the mucosal immune system, and the mucosal epithelium. The stability of gut flora depends on intestinal barrier function [ 7 ]. Inflammatory responses, bacterial translocation, and impaired intestinal barrier are essential for AP progression and complications [ 8 , 9 ]. Escherichia coli , Enterococcus , and Enterobacteriaceae are the primary pathogens that cause secondary infections in AP [ 10 , 11 ]. Additionally, dysbiosis of the intestinal flora causes a decrease in the levels of short-chain fatty acid-producing bacteria, which affects the integrity of the intestinal barrier and worsens clinical conditions [ 12 ]. Although previous studies have reported potential routes and mechanisms for the development of AP, more information on intestinal dysfunction over the entire course of AP is required, especially during the recovery phase [ 13 ]. In this study, we employed metagenomic sequencing technology to investigate the gut microbiota composition and function in patients with AP of varying severities during the acute and recovery phases, revealing the complex interaction between gut microbes and the host. Material and Methods Patients From January 2021 to September 2021, patients diagnosed based on the 2012 Revision of the Atlanta Classification and admitted to the Peking Union Medical College Hospital, Beijing, China, were included in this study. Patients were enroled within 48 h of disease onset and followed up for 30 d to observe their recovery. Patients were excluded if they had a history of immunodeficiency, inflammatory bowel disease, specificity, asthma, irritable bowel syndrome, celiac disease, gastroenteritis, colon cancer, human immunodeficiency virus infection, necrotising enterocolitis, or arthritis. The criteria for individuals recovering from AP were as follows: (1) relief of abdominal pain in patients with mild AP (MAP); (2) decrease in the improved Marshall score to 0 points in patients with moderately severe AP (MSAP) or severe AP (SAP); and (3) tolerable enteral nutrition. The control group was defined as described in our previous study [10]. Four healthy individuals were recruited and matched for age, sex, and body mass index. Written informed consent was obtained from all the patients. Approval was obtained from the Institutional Review Board of Peking Union Medical College Hospital (No. JS1826). Sample collection A flowchart of the study is shown in Figure S1. Rectal swabs were obtained from each patient with AP immediately after admission and during the recovery phase, owing to the difficulties in direct fecal collection due to gastrointestinal dysfunction [14, 15]. The control group consisted of four healthy volunteers, with all individuals having empty stomachs before sample collection. All individuals were enroled at Peking Union Medical College Hospital, Beijing, China. Fecal sample collection methods have been previously described in detail [10]. Soap, water, and 70% alcohol were used to remove the anus. The disinfectant was evaporated independently to reduce commensal skin contamination. The sterile swab was infiltrated with normal saline for 2 min before being inserted into the anus as deep as 4–5 cm and then rotated gently to obtain fecal samples. Rectal swabs with fecal samples were quickly placed into a sterile tube and immediately stored at -80℃ before shipping to the laboratory under cold chain. Metagenomics sequencing, quality control, and genome assembly DNA was extracted from the fecal samples according to the manufacturer’s instructions. DNA purity, concentration, and quality were determined using NanoDrop2000 (Thermo Fisher Scientific, Waltham, MA, USA), TBS-380, and a 1% agarose gel electrophoresis system, respectively. Paired-end sequencing was performed on an Illumina HiSeq4000 platform (Illumina, San Diego, CA, USA). After removing the low-quality reads, the reads were aligned to the human genome using BWA (v 0.7.9a) [16]. After quality control, the reads were mapped to representative genes with 95% identity using SOAPaligner (v 2.21, http://soap.genomics.org.cn/) [17], and gene abundance was assessed for each sample. Alpha diversity and principal component analysis α-diversity analysis was performed using the R packages “vegan” (v 4.0.5) and “reshape2” (v 4.0.5). Species diversity was assessed using Shannon’s index, with a higher value indicating higher floral diversity. In addition, principal component analysis, which reflects the differences and distances between samples by analysing the community composition of different samples, was performed. The relatively similar mean values of the samples from different groups indicated a similarity in species composition. Analysis of typing and dominant microbiome composition Microbiome typing analysis involves typing of the dominant microflora structures in different samples using statistical clustering. Through this method, different samples with structures similar to those of the dominant microbiome can be grouped into one class. This method is mainly applied to the specific environmental samples. Based on the relative abundance of the microbiome at the genus level, the Bray–Curtis distance was calculated for clustering. After calculating the optimal clustering K using the Calinski–Harabasz index, the 28 samples were divided into different types. In patients with AP and those recovering from AP, a Circos diagram was used to visualise the distribution of the dominant microflora among different samples. Differential microbiome analysis and functional annotation Based on the read abundance data, species with significant differences were identified at P < 0.05, using the Wilcoxon rank-sum test. Nonredundant genes were annotated against the KEGG database (v 94.2, http://www.genome.jp/keeg/) [18] using Diamond (v 2.0.13, http://ab.inf.uni-tuebingen.de/software/diamond/) [19] at an optimised e-value (cutoff of 1e − 5). Statistical analyses Statistical analyses and data visualisation were conducted using custom scripts that are publicly available (https://cloud.majorbio.com/page/project/task.html?project_id=7df904c5bhnc1vgo2oj2fkevfr) [20]. The Strengthening The Organizing and Reporting of Microbiome Studies checklist was completed and uploaded to Zenodo (https://zenodo.org/records/10676912?token=eyJhbGciOiJIUzUxMiJ9.eyJpZCI6ImM1YTIzYmI0LTU0MmYtNDQ4NS05NzdkLTVmOTRkOWFmNGI0NCIsImRhdGEiOnt9LCJy YW5kb20iOiIxNjE4NmI1NWVmNjc1ZTY4YjYyMjcxOTRkNTI5NzRlMyJ9.T3S5fNFyjdfxPgK-BGoCYPaVx4H7gKLMLGcJ8cYuogIp9mIhJkdl1QUqH9vJm5qaEeZmEmue2Jqqc1XCFLo28A). Results Patient characteristics Twelve patients with AP and four healthy controls were included in this study. Among the 12 patients with AP, 4 had MAP, 5 had MSAP, and 3 had SAP. After treatment, the clinical condition of all patients with AP initially improved and their serum lipase and amylase levels recovered or met the expected values. Meanwhile, APACHE II and Balthazar CT scores decreased during the recovery phase. Detailed information on the patients with AP and healthy controls is presented in Table S1 . Gut microbiome diversity in patients with AP during acute and recovery phases Compared with that in healthy controls, the α-diversity (Shannon index) in patients with AP was significantly reduced during the acute phase and further decreased during the recovery phase, despite improvement of clinical manifestations (Fig. 1 A). A reduction in diversity was observed among different severities during the recovery phase (Fig. 1 B). During the recovery period, clinical manifestations started to improve but gut microbiota diversity did not recover concomitantly. Thus, the recovery of gut microbiota and intestinal function was slower than the overall clinical improvement. Category-based principal coordinate analysis (PCoA) revealed differences in β-diversity between the control and AP groups, showing distinct clustering. However, no significant clustering was observed between the acute and recovery phases or among the MAP, MSAP, and SAP subgroups (Fig. 1 C). The structural diversity of acute-phase samples only slightly differed at different severity levels (Figure S2 ). During the recovery phase, the samples with different severities exhibited clear clustering. Analysis of enterotypes and dominant microbiome composition during the recovery phase of AP The 28 samples were clustered into two distinct enterotypes, with those having a high abundance of the Bacteroides genus being classified as enterotype 1 and those having a high abundance of the Enterococcus genus classified as enterotype 2 for the total group (Fig. 2 A) and subgroups (Fig. 2 B). Enterococcus was the dominant genus in the gut microbiota during the acute and recovery phases of AP (Fig. 2 C). At the species level, the abundances of unclassified_g__Enterococcus and Enterococcus_faecium were high during the acute and recovery phases of AP, which is in accordance with the results at the genus level (Fig. 2 D). Similarly, in this subgroup, Enterococcus was the dominant genus in the gut microbiota during the acute and recovery phases of AP (Fig. 2 E). At the species level, the abundances of unclassified_g__Enterococcus and Enterococcus_faecium were high during the acute and recovery phases of AP, which is in accordance with the results at the genus level (Fig. 2 F). Comparison of the acute and recovery phases of MAP revealed a decrease in the abundance of Enterococcus (Figure S3 A) and an increase in that of Bacteroidales (Figure S3 B) increased (without significant differences). Conversely, comparison of the acute and recovery phases of MSAP revealed an increase in the abundance of Enterococcus (Figure S3 C) and a decrease in that of Bacteroidales (Figure S3 D) (without significant differences). Similarly, comparison of the acute and recovery phases of SAP revealed an increase in the abundance of Enterococcus (Figure S3 E) and a decrease in that of Bacteroidales (Figure S3 F) (without significant differences). Thus, Enterococcus was the dominant genus of the gut microbiota during recovery. Differential microbiome analysis during the recovery phase of AP Differential species composition analysis showed that unclassified_g__Enterococcus was the most abundant species during the acute phase of AP (Fig. 3 A). During the recovery phase, the abundance of beneficial bacteria such as Faecalibacterium_prausnitzii , Bifidobacterium_longum , and Firmicutes_bacterium was low, indicating that the overall abundance of beneficial bacteria had not recovered during this period (Fig. 3 B). At the second level (90 species of bacteria in the acute and recovery phases of AP) (Fig. 3 C), 29 species showed an inverse trend in abundance during the acute and recovery phases in patients with AP (Table 1 ). The short-chain fatty acid-producing bacteria Anaerococcus , Blautia , Candidatus_Blautia_pullistercoris , and Eubacterium_sp._CAG:86 were low in abundance during the recovery phase. The abundance of the propionic and butyric acid-producing bacterium Roseburia_inulinivorans_CAG:15 was low during the acute phase of AP, whereas that of Candidatus_Eisenbergiella_pullicola was high. The levels of anti-inflammatory microbiota began to increase during the recovery phase; however, those of other beneficial bacteria did not return to normal. Table 1 The 29 kinds of bacteria showed a reverse trend in abundance levels between the acute and recovery phases of AP patients Species Acute phase vs Control Recovery phase vs Acute phase s__unclassified_g__Anaerococcus up down s__Beta_vulgaris up down s__Alistipes_senegalensis up down s__Paludibacteraceae_bacterium down up s__Blautia_sp._CAG:52 up down s__Cajanus_cajan up down s__Roseburia_inulinivorans_CAG:15 down up s__Pelagicola_marinus up down s__Corynebacterium_urealyticum down up s__Pseudoramibacter_alactolyticus down up s__Trifolium_pratense up down s__Helianthus_annuus up down s__Dorea_longicatena_CAG:42 up down s__Oryza_sativa up down s__Dorcoceras_hygrometricum up down s__Candidatus_Blautia_pullistercoris up down s__Juglans_regia up down s__Candidatus_Eisenbergiella_pullicola up down s__Rosa_chinensis up down s__Hevea_brasiliensis up down s__bacterium_1XD8-92 up down s__Diploscapter_pachys down up s__Fallopia_multiflora up down s__Eubacterium_sp._CAG:86 up down s__Corynebacterium_massiliense down up s__Panicum_virgatum up down s__Clostridium_sp._CCUG_7971 up down s__Pseudoglutamicibacter_albus down up s__Methanobrevibacter_oralis up down Comparison of the number of species between patients with MAP and healthy controls revealed 498 significantly different species of gut microbiota. The levels of Enterococcus and Bacteroidales significantly increased and decreased, respectively (Fig. 4 A). Comparison of the acute and recovery phases of MAP revealed 13 significantly different species of gut microbiota. The abundances of Oscillibacter , Ruminococcus , and Firmicutes significantly decreased during the recovery phase (Fig. 4 B). Comparison of patients with MSAP and healthy controls revealed 1223 significantly different species of gut microbiota. Enterococcus was the most significantly increased genus in the gut microbiota (Fig. 4 C). In the MSAP group, 241 significantly different species were identified between the recovery and acute phases. Adlercreutzia equolifaciens showed the most significant decrease in abundance; however, its abundance was low during the recovery phase (Fig. 4 D). Comparison of patients with SAP and healthy controls revealed 397 significantly different species of gut microbiota. The abundances of Enterococcus and Porphyromonas asaccharolytica significantly increased and decreased, respectively (Figure S4 A). No significant differences in gut microbiota species between the acute and recovery phases of SAP were found (Figure S4 B). Our results suggest that patients with SAP may have recovered from their clinical symptoms; however, their microbiota profiles have not yet recovered. Enterococcus was the most abundant genus in the acute phase of AP at varying severities. Two common species were identified between the 498 differential species during the acute phase of MAP and 13 differential species during the recovery phase of MAP (Fig. 4 E). Firmicutes_bacterium_CAG:129_59_24 , belonging to Firmicutes, showed low abundance during the recovery phase of MAP, indicating that its function had not yet recovered. Intestinimonas_timonensis , a pro-inflammatory microorganism, showed decreased abundance during the recovery phase of MAP, indicating that the inflammatory function of the species gradually recovered. Between the 1223 differential species during the acute phase of MSAP and 241 differential species during the recovery phase, 47 common species were identified (Fig. 4 F). Of these, 19 were highly abundant in patients with MSAP during the acute phase but showed low abundance during the recovery phase (Table 2 ). Among them, Candidatus_Eisenbergiella_pullicola and Collinsella_provencensis , which are related to intestinal wall permeability, Olsenella_sp._Marseille-P4518 , which is a fructose-consuming strain, Clostridium_sp._D53t1_180928_C8 , Blautia_sp._AM28-10 , and Olsenella_sp._Marseille-P4518 were highly abundant during the acute phase of MSAP but decreased in abundance during the recovery phase. Table 2 The 19 kinds of bacteria showed a reverse trend in abundance levels between the acute and recovery phase of MASP patients Species A_MSAP vs Control R_MSAP vs A_MSAP s__Candidatus_Eisenbergiella_pullicola up down s__Prunus_dulcis up down s__Hevea_brasiliensis up down s__Myoviridae_sp._ct3Oc10 up down s__Arachis_hypogaea up down s__Fallopia_multiflora up down s__Trema_orientale up down s__Collinsella_provencensis up down s__Candidatus_Aphodovivens_avistercoris up down s__Clostridium_sp._CCUG_7971 up down s__Candidatus_Blautia_pullistercoris up down s__Olsenella_sp._Marseille-P4518 up down s__Pogostemon_cablin up down s__Betaproteobacteria_bacterium up down s__Clostridium_sp._D43t1_170807_H7 up down s__Blautia_sp._AM28-10 up down s__Turicibacter_sp._H121 up down s__Methanobrevibacter_oralis up down s__Clostridium_sp._D53t1_180928_C8 up down A_MSAP, acute phase of MSAP; R_MSAP, recovery phase of MSAP. Analysis of differences in functional compositions in the recovery phase of AP The gut microbiota of patients with acute-phase AP was involved in 87 significantly different functional compositions of the KEGG pathway. Adenosine triphosphate-binding cassette (ABC) transporters, fructose and mannose metabolism, and activation of the phosphotransferase system (PTS) were significantly upregulated, whereas glycine, serine, and threonine metabolism was significantly downregulated during the acute phase of AP (Fig. 5 A). Meanwhile, 13 significantly different functional pathways were identified during the recovery phase, of which microbial metabolism was significantly activated in diverse environments. However, RNA degradation, protein processing in the endoplasmic reticulum, ribosome biogenesis in eukaryotes, and other RNA- and protein-related pathways were inhibited or downregulated (Fig. 5 B). Only three pathways were shared between acute and recovery phases: RNA degradation, flavonoid biosynthesis, and stilbenoid, diarylheptanoid, and gingerol biosynthesis (Fig. 5 C). The changes in pathway function for \ patients with varying AP severities are shown in Fig. 6 and summarised in Table 3 . We identified 37, 98, and 23 significantly different KEGG functional compositions in patients with MAP, MSAP, and SAP, respectively. Table 3 KEGG functional composition difference analysis of the microbiota in the recovery phase of AP with different severity. Acute phase Recovery phase MAP Phosphotransferase System (PTS) ↑ Glycerolipid Metabolism ↑ Glycine, serine, and threonine metabolism ↓ Glyoxylate and dicarboxylate metabolism ↓ Citrate cycle (TCA cycle) ↓ Butanoate metabolism ↓ Cardiac muscle contraction ↓ mTOR signaling pathway ↓ Phagosome ↓ MSAP ABC transporters ↑ Phosphotransferase system (PTS) ↑ Fructose and mannose metabolism ↑ Carbon metabolism ↓ Amino sugar and nucleotide sugar metabolism ↓ Glycine, serine, and threonine metabolism ↓ None SAP Nucleocytoplasmic transport ↑ Steroid biosynthesis ↑ Inflammatory mediator regulation of TRP channels ↑ Viral myocarditis ↑ None Discussion The onset, development, and recovery of AP are closely related to intestinal flora dysbiosis and bacterial translocation [ 21 ]. The intestinal flora can participate in the onset and recovery of AP by influencing host metabolism and intestinal mucosal permeability [ 22 ]. We performed shotgun metagenomic sequencing on 12 patients with AP during the acute and recovery phases. Significant microbiome dysbiosis was observed during both phases, suggesting an association between dysbiosis and recovery from AP. Enterococcus , a harmful bacterium, was the most abundant genus present during the acute and recovery phases of AP. For example, the relative abundances of s__Enterococcus_faecium , s__Enterococcus_faecalis , s__Enterococcus_hirae , s__Enterococcus_gallinarum , and s__Enterococcus_casseliflavus were high during the acute phase of AP. Enterococcus can attach to and penetrate host cells, traverse host epithelial barriers, and gain entry into the systemic circulation and other organs, promoting its spread within the host [ 12 ]. The relative abundance of Enterococcus spp. is associated with infection and systemic inflammation [ 23 ]. Biofilm formation by Enterococcus involves a complex interplay between genes and virulence factors, including gelatinases, cytolysins, secretory antigen A, fimbriae, microbial surface components that recognise adhesion matrix molecules, and DNA [ 24 ]. Zhang et al. reported that the relative abundance of the potentially pathogenic genus Enterococcus increased in the SAP group but decreased after Qingyi decoction treatment [ 25 ]. As a 0000000, Enterococcus faecium performed well in distinguishing patients with acute necrotising pancreatitis from those with non-necrotising pancreatitis [ 26 ]. In patients with Crohn's disease, ulcerative colitis, or ischemic colitis, the abundance of potentially pathogenic bacteria, such as Enterococcus faecium and Enterococcus faecalis , is increased, indicating their potential diagnostic value [ 27 ]. During the recovery phase of AP, the abundances of the beneficial bacteria Faecalibacterium_prausnitzii , Bifidobacterium_longum , and Firmicutes_bacterium were significantly lower than those during the acute phase. These results indicate that the clinical manifestations began to improve, but the overall abundance of beneficial bacteria did not recover concomitantly during the AP recovery period. Recovery of the gut microbiota and intestinal function was slower than the overall clinical improvement. Faecalibacterium_prausnitzii is involved in inflammatory processes, blocking nuclear factor kappa B activation and interleukin (IL)-8 production [ 28 ]. The abundance of Faecalibacterium prausnitzii was lower in patients with chronic pancreatitis (CP) and diabetes than in those without diabetes and in controls [ 29 ]. Similarly, McEachron et al.. observed a significantly decreased abundance of Faecalibacterium in patients with CP [ 30 ]. Bifidobacterium has been reported to reduce the risk of multiple organ failure in SAP [ 31 ]. Bifidobacteria , with reduced abundance in feces, is inversely correlated with the severity of the systemic inflammatory response in patients with AP [ 32 ]. Firmicutes , which are pathogenic Gram-positive bacteria, are mainly butyrate producers and have been identified in patients with AP [ 33 ]. Anaerococcus , Blautia , Candidatus_Blautia_pullistercoris , Eubacterium_sp._CAG:86 , and other short-chain fatty acid-producing bacteria were significantly lower in abundance during the recovery phase of AP. In our previous study, we observed a significantly increased abundance of Anaerococcus in MSAP and a significantly decreased abundance of Blautia in MAP [ 10 ]. The abundance of the propionic and butyric acid-producing bacterium Roseburia_inulinivorans_CAG:15 was significantly lower during the recovery phase, whereas that of Candidatus_Eisenbergiella_pullicola was significantly higher during the acute phase of AP. Faecalibacterium and Roseburia are well-known butyrate producers that attenuate intestinal inflammation and are beneficial to the host [ 34 , 35 ]. The relative abundance of Roseburia decreased in mice with CP [ 36 ]. Eisenbergiella has been suggested to protect mice with ulcerative colitis by reducing the intestinal inflammatory response [ 37 ]. Bacterial abundance decreased significantly during the recovery phase, indicating an improvement in inflammation-related microbiota function. However, the abundance of beneficial bacteria did not fully recover during the AP recovery phase. In MAP, the abundance of Oscillibacter_sp. significantly increased during the acute phase but decreased during the recovery phase. Increased abundance of Oscillibacter is associated with intestinal permeability and host inflammation in patients with large-artery atherosclerotic stroke or transient ischemic attacks [ 38 ]. Oscillibacter sp. 57_20 has been reported to identify SAP at an early stage [ 22 ]. In MSAP, the abundances of Clostridium_sp._D53t1_180928_C8 and Blautia_sp._AM28-10 significantly increased during the acute phase but decreased during the recovery phase. Clostridium perfringens was isolated from cultures of pancreatic tissue obtained from a patient with AP during hospitalisation [ 39 ]. Significant differences in Collinsella abundance were observed between the MAP and SAP groups [ 40 ]. The abundance of Collinsella is associated with the production of the proinflammatory cytokine IL-17A [ 41 ]. Decreased abundance of Collinsella has been reported in children with CP [ 42 ]. In addition, Candidatus_Eisenbergiella_pullicola and Collinsella_provencensis were associated with intestinal wall permeability. In SAP, the abundance of Adlercreutzia_equolifaciens was high during the acute phase but decreased during recovery. However, no significant difference in its abundance was found between the two phases, suggesting a possible intrinsic role for SAP. Additionally, we observed that disturbances in gut microbiota affect host metabolism and molecular transport mechanisms. PTS was significantly activated, whereas glycine, serine, and threonine metabolism was significantly inhibited during the acute phase of AP. PTS, one of the most efficient sugar transport systems, plays a crucial role in the regulation of glucose metabolism in bacteria [ 43 ]. Compared with their levels 6 weeks after hospital discharge, the levels of glycine, serine, and threonine in patients with AP were significantly lower upon admission [ 44 ]. Glycine administration improves AP pathomorphology and reduces the severity of MAP and SAP [ 45 ]. During the AP recovery phase, microbial metabolism is significantly activated and upregulated in diverse environmental pathways. However, RNA degradation, protein processing in the endoplasmic reticulum, ribosome biogenesis in eukaryotes, and other functional pathways related to RNA and proteins were inhibited and downregulated. These findings indicate that while intestinal function and the gut microbiota structure start to recover, the restoration of RNA- and protein-related biological processes lags behind. During the recovery phase of MAP, an increase in the functional composition of glycine, serine, and threonine metabolism and a decrease in the functional composition of the PTS compared with the acute phase was observed. In addition, cardiac muscle contraction was the most significantly reduced functional component during the MAP recovery phase. The PTS regulates carbohydrate uptake and is considered a potential biomarker of inflammatory bowel disease [ 46 ]. Glycine, serine, and threonine metabolism is involved in glycolytic biosynthesis in pancreatic cancer [ 47 ]. Regardless of its severity, AP is accompanied by cardiac injury, and the degree of damage is associated with the clinical type of AP [ 48 ]. Thus, activation of the glycine, serine, and threonine metabolic pathways, deactivation of the phosphotransferase system, and reduction of cardiac muscle contraction are associated with the recovery phase in patients with MAP. Moreover, during the recovery phase of MSAP, an increase in the functional composition of carbon metabolism and a decrease in the functional composition of ABC transporters compared with the acute phase was observed. ABC transporters translocate various endogenous metabolites and cytotoxic compounds across the lipid bilayer [ 49 ]. In cancer, overexpression of ABC transporters leads to drug resistance [ 50 ]. In mice with AP, antioxidant treatment with carbon monoxide-bound hemoglobin vesicles effectively reduced the inflammatory response and pancreatic tissue damage [ 51 ]. Therefore, carbon metabolism and ABC transporters may be involved in MSAP recovery. These signalling pathways exhibit an opposite trend during the recovery phase of MAP and MSAP in comparison with the acute phase, suggesting that they may benefit recovery. However, this study had several limitations. The sample size used in shotgun metagenomics was relatively small, and further studies with more participants are warranted. In addition, the causal links among the microbiota, functional composition, and AP recovery require further investigation. In conclusion, we identified the presence of several bacteria, along with their functional composition, during the recovery phase of AP through a shotgun metagenomic survey, extending the current understanding of gut microbiota during this phase. This study provides information that may guide the development of treatment strategies and the prognosis of AP. Abbreviations AP, Acute pancreatitis MAP, mild AP MSAP, moderately severe AP SAP, severe AP PCoA, principal coordinate analysis ABC, Adenosine triphosphate-binding cassette PTS, phosphotransferase system CP, chronic pancreatitis Declarations Ethics approval and consent to participate All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional review board of the Peking Union Medical College Hospital (No. JS1826) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study. Informed consent was obtained from all individual participants included in the study. Consent for publication Not Applicable. Data availability The raw data had been deposited in the NCBI Sequence Read Archive (BioProject accession ID: PRJNA1074432, http://www.ncbi.nlm.nih.gov/bioproject/1074432). The full results of all analyses were included in Table S2-5. Funding This study was funded by the National Natural Science Foundation of China (grant number 32170788), the National High Level Hospital Clinical Research Funding (grant number 2022-PUMCH-B-023), the National Key Clinical Specialty Construction Project (grant number ZK108000), and Natural Science Foundation of Beijing (grant number 7232123). Competing interests The authors declare that they have no conflict of interest. Acknowledgments We extend our sincere gratitude to Taylor & Francis Editing Services for their meticulous editing and valuable contributions in enhancing the quality and clarity of our manuscript. Authors’ contributions X.S., J.N.L., D.W.* and Q.W.: study design, interpretation of data, and drafting of the manuscript. X.S., J.N.L., and D.W. † contributed equally to this work and are co-first authors. Z.Y.H., X.X.Y., and Z.H.Y.: sample collection, statistical analysis, and critical revision of the manuscript. J.X., Q.W. and D.W.#: study concept and critical revision. D.W.#: final approval of the manuscript. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. References Gukovskaya, A. S. et al. Recent Insights Into the Pathogenic Mechanism of Pancreatitis: Role of Acinar Cell Organelle Disorders. Pancreas . 48, 459-470 (2019). Hammer, H. F. 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The role of ATP-binding cassette (ABC) proteins in protozoan parasites. Mol Biochem Parasitol . 167, 81-94 (2009). Fletcher, J. I., Haber, M., Henderson, M. J. & Norris, M. D. ABC transporters in cancer: more than just drug efflux pumps. Nat Rev Cancer . 10, 147-156 (2010). Nagao, S. et al. Carbon monoxide-bound hemoglobin vesicles ameliorate multiorgan injuries induced by severe acute pancreatitis in mice by their anti-inflammatory and antioxidant properties. Int J Nanomedicine . 11, 5611-5620 (2016). Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigures.pdf Supplemental information Figure S1 The flow chart of this study Figure S2 PCoA of Bray-Curtis distance analysis in the AP patients of different severity A_MAP, acute phase of MAP; R_MAP, recovery phase of MAP; A_MSAP, acute phase of MSAP; R_MSAP, recovery phase of MSAP; A_SAP, acute phase of SAP; R_SAP, recovery phase of SAP. Figure S3 Difference analysis of Enterococcus and Bacteroidales in the acute phase of MAP and recovery phase of MSAP A: Difference analysis of Enterococcus in MAP; B: difference analysis of Bacteroidales in MAP. C: difference analysis of Enterococcus in MSAP; D: difference analysis of Bacteroidetes in MSAP; E: difference analysis of Enterococcus in SAP; F: difference analysis of Bacteroidetes in SAP. A_MAP, acute phase of MAP; R_MAP, recovery phase of MAP; A_MSAP, acute phase of MSAP; R_MSAP, recovery phase of MSAP; A_SAP, acute phase of SAP; R_SAP, recovery phase of SAP. *0.01<p≤0.05, ns: not significant. Figure S4 Difference analysis of gut microbiota species in the recovery phase of SAP A: Difference analysis of gut microbiota species in SAP vs. CON; B: difference analysis of gut microbiota species in the recovery phase of SAP vs. SAP. A_SAP, acute phase of SAP; R_SAP, recovery phase of SAP. *0.01<p≤0.05. TableS1ClinicalinformationofenrolledhealthycontrolsandAPpatients.xlsx Table S1 Clinical information of enrolled healthy controls and AP patients. MAP, mild acute pancreatitis; MSAP, moderately severe acute pancreatitis; SAP, severe acute pancreatitis. TableS2DifferenceanalysisofgutmicrobiotaspeciesintherecoveryphaseofAP.xlsx Table S2 Difference analysis of gut microbiota species in the recovery phase of AP. TableS3DifferenceanalysisofgutmicrobiotaspeciesintherecoveryphaseofAPwithdifferentseverity.xlsx Table S3 Difference analysis of gut microbiota species in the recovery phase of AP with different severity. TableS4KEGGfunctionalcompositiondifferenceanalysisofthemicrobiotaintherecoveryphaseofAP.xlsx Table S4 KEGG functional composition difference analysis of the microbiota in the recovery phase of AP. TableS5KEGGfunctionalcompositiondifferenceanalysisofthemicrobiotaintherecoveryphaseofAPwithdifferentseverity.xlsx Table S5 KEGG functional composition difference analysis of the microbiota in the recovery phase of AP with different severity. Cite Share Download PDF Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 13 Jan, 2025 Reviews received at journal 11 Jan, 2025 Reviews received at journal 24 Dec, 2024 Reviewers agreed at journal 22 Dec, 2024 Reviewers agreed at journal 20 Dec, 2024 Reviewers invited by journal 20 Dec, 2024 Editor assigned by journal 12 Dec, 2024 Editor invited by journal 18 Nov, 2024 Submission checks completed at journal 17 Nov, 2024 First submitted to journal 14 Nov, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5453055","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":389943413,"identity":"7209b283-a6c0-4df1-b40c-594f5a6608e1","order_by":0,"name":"Xiao Song","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Song","suffix":""},{"id":389943414,"identity":"40794c11-d31d-44f7-ac0f-1cf91aa0b0ca","order_by":1,"name":"Jia-Ning Li","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Jia-Ning","middleName":"","lastName":"Li","suffix":""},{"id":389943415,"identity":"924faaaa-571a-45fa-8f0c-d8003ac1df43","order_by":2,"name":"Duan Wang","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Duan","middleName":"","lastName":"Wang","suffix":""},{"id":389943416,"identity":"2ded1e57-cd12-41e7-8558-9d80da3be7a0","order_by":3,"name":"Zi-Ying Han","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Zi-Ying","middleName":"","lastName":"Han","suffix":""},{"id":389943417,"identity":"5e0345e8-138c-4fac-9c5c-33b43e8fcc89","order_by":4,"name":"Xia-Xiao Yan","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Xia-Xiao","middleName":"","lastName":"Yan","suffix":""},{"id":389943418,"identity":"c760e237-a8d9-4b19-ac7a-e3213e3155e0","order_by":5,"name":"Zi-Han Yang","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Zi-Han","middleName":"","lastName":"Yang","suffix":""},{"id":389943419,"identity":"efc25ffe-3071-4757-a5a9-d7999a5af954","order_by":6,"name":"Jun Xu","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Xu","suffix":""},{"id":389943420,"identity":"29bbf11a-ccc2-4da8-ba21-c13146447f41","order_by":7,"name":"Qiang Wang","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Qiang","middleName":"","lastName":"Wang","suffix":""},{"id":389943421,"identity":"8d24a3a7-c425-434e-8e6a-3a110ca10155","order_by":8,"name":"Dong Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAoElEQVRIiWNgGAWjYHACAyC2ATHYSNKSRrqWwyRoMbiRvPFxwa/zeQbXDj97wFBzhxgtacXGM/tuFxvcTjM3YDj2jLAWsxs5ZtK8PbcTN9zOYZNgbDhMtJZzpGrh+XGABC32Z54VG/M2JBdL3k4zk0g4RoQWyXZgiPH8scvju538TOJDDRFawICxjSEBzEggUgMQ/CFF8SgYBaNgFIw4AACwCTzCUdveZQAAAABJRU5ErkJggg==","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":true,"prefix":"","firstName":"Dong","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2024-11-14 10:23:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5453055/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5453055/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-05127-5","type":"published","date":"2025-07-01T15:56:54+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":71876443,"identity":"c0e0534d-0d7b-44cb-8577-4177419c40b3","added_by":"auto","created_at":"2024-12-19 11:00:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":324734,"visible":true,"origin":"","legend":"\u003cp\u003eMicrobial diversity and principal component analysis in the recovery phase of AP.\u003c/p\u003e\n\u003cp\u003eA: Alpha diversity analysis in acute and recovery phases of AP patients; B: Alpha diversity analysis in the AP patients of different severity; C: PCoA of Bray-Curtis distance analysis in all samples based on species level. A_MAP, acute phase of MAP; R_MAP, recovery phase of MAP; A_MSAP, acute phase of MSAP; R_MSAP, recovery phase of MSAP; A_SAP, acute phase of SAP; R_SAP, recovery phase of SAP.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5453055/v1/a58c6567ead94ad77ad3ccdf.png"},{"id":71876970,"identity":"ba977f7f-a359-4c6f-91d0-c691d32c5386","added_by":"auto","created_at":"2024-12-19 11:08:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2090585,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of enterotypes and dominant microbiome composition in the recovery phase of AP.\u003c/p\u003e\n\u003cp\u003eA: Analysis of typing in the subgroup; B: analysis of typing in the total group. Distribution of the predominant dominant gut microbiota species at the genus level in total group (C), at the species level in total group (D), at the genus level in the subgroup (E), and at the species level in subgroup (F). A_MAP, acute phase of MAP; R_MAP, recovery phase of MAP; A_MSAP, acute phase of MSAP; R_MSAP, recovery phase of MSAP; A_SAP, acute phase of SAP; R_SAP, recovery phase of SAP.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5453055/v1/88610d4d154e28146ec93a7a.png"},{"id":71876447,"identity":"1bfdecc7-b8fb-4ce5-9c25-d031c0cc74cc","added_by":"auto","created_at":"2024-12-19 11:00:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":473138,"visible":true,"origin":"","legend":"\u003cp\u003eDifference analysis of gut microbiota species in the recovery phase of AP.\u003c/p\u003e\n\u003cp\u003eA: Difference analysis of gut microbiota species in AP vs. Control; B: difference analysis of gut microbiota species in the recovery phase of AP vs. acute phase of AP; C: Venn plot of gut microbiota species in the total group. A, acute phase; R, recovery phase. *0.01\u0026lt;p≤0.05, ns: not significant.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5453055/v1/886fa08f869ea696925a251c.png"},{"id":71876444,"identity":"c0e69460-d9e5-4be2-ad9c-f1680cf945d1","added_by":"auto","created_at":"2024-12-19 11:00:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1504190,"visible":true,"origin":"","legend":"\u003cp\u003eDifference analysis of gut microbiota species in the recovery phase of MAP and MSAP.\u003c/p\u003e\n\u003cp\u003eA: Difference analysis of gut microbiota species in MAP vs. Control; B: difference analysis of gut microbiota species in the recovery phase of MAP vs. acute phase of MAP; C: difference analysis of gut microbiota species in MSAP vs. Control; D: difference analysis of gut microbiota species in the recovery phase of MSAP vs. acute phase of MSAP; E: Venn plot of gut microbiota species in MAP; F: Venn plot of gut microbiota species in MSAP. A_MAP, acute phase of MAP; R_MAP, recovery phase of MAP; A_MSAP, acute phase of MSAP; R_MSAP, recovery phase of MSAP. *0.01\u0026lt;p≤0.05, ns: not significant.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5453055/v1/c62c291fa9a5d91fc23f6d05.png"},{"id":71876971,"identity":"13290501-6da6-4ba1-af5b-36a2f6b82674","added_by":"auto","created_at":"2024-12-19 11:08:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":443526,"visible":true,"origin":"","legend":"\u003cp\u003eKEGG functional composition difference analysis of the microbiota in the recovery phase of AP.\u003c/p\u003e\n\u003cp\u003eA: Difference analysis of functional compositions of KEGG in AP vs. Control; B: difference analysis of functional compositions of KEGG in the recovery phase of AP vs. acute phase of AP; C: Venn plot of functional compositions in AP. A, acute phase; R, recovery phase. *0.01\u0026lt;p≤0.05.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5453055/v1/f7db5ed5e69fe62920c13eba.png"},{"id":71879624,"identity":"5a34cacd-7821-492b-8c8c-9b1003c445e6","added_by":"auto","created_at":"2024-12-19 11:24:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1161417,"visible":true,"origin":"","legend":"\u003cp\u003eKEGG functional composition difference analysis of the microbiota in the recovery phase of AP with different severity.\u003c/p\u003e\n\u003cp\u003eA: difference analysis of functional compositions of KEGG in MAP vs. Control; B: difference analysis of functional compositions of KEGG in recovery phase of MAP vs. acute phase of MAP; C: difference analysis of functional compositions of KEGG in MSAP vs. Control; D: difference analysis of functional compositions of KEGG in recovery phase of MSAP vs. acute phase of MSAP; E: difference analysis of functional compositions of KEGG in SAP vs. Control; F: difference analysis of functional compositions of KEGG in recovery phase of SAP vs. acute phase of SAP. A_MAP, acute phase of MAP; R_MAP, recovery phase of MAP; A_MSAP, acute phase of MSAP; R_MSAP, recovery phase of MSAP; A_SAP, acute phase of SAP; R_SAP, recovery phase of SAP. *0.01\u0026lt;p≤0.05.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5453055/v1/58ab371ac8f7a93040c494ff.png"},{"id":86178876,"identity":"5a8f7244-e228-4ac9-85b7-50558a0a7cf8","added_by":"auto","created_at":"2025-07-07 16:06:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6403666,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5453055/v1/0eb210c9-a034-4965-b16b-8ee4065e3eca.pdf"},{"id":71876452,"identity":"96ad0e24-8c45-47d6-a857-890feb424234","added_by":"auto","created_at":"2024-12-19 11:00:03","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":957757,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S1\u003c/strong\u003e The flow chart of this study\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S2\u003c/strong\u003e PCoA of Bray-Curtis distance analysis in the AP patients of different severity\u003c/p\u003e\n\u003cp\u003eA_MAP, acute phase of MAP; R_MAP, recovery phase of MAP; A_MSAP, acute phase of MSAP; R_MSAP, recovery phase of MSAP; A_SAP, acute phase of SAP; R_SAP, recovery phase of SAP.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S3\u003c/strong\u003e Difference analysis of\u003cem\u003e Enterococcus\u003c/em\u003e and \u003cem\u003eBacteroidales\u003c/em\u003e in the acute phase of MAP and recovery phase of MSAP\u003c/p\u003e\n\u003cp\u003eA: Difference analysis of\u003cem\u003e Enterococcus\u003c/em\u003e in MAP; B: difference analysis of\u003cem\u003e Bacteroidales\u003c/em\u003e in MAP. C: difference analysis of\u003cem\u003e Enterococcus\u003c/em\u003e in MSAP; D: difference analysis of\u003cem\u003eBacteroidetes\u003c/em\u003e in MSAP; E: difference analysis of\u003cem\u003e Enterococcus\u003c/em\u003e in SAP; F: difference analysis of\u003cem\u003e Bacteroidetes\u003c/em\u003e in SAP. A_MAP, acute phase of MAP; R_MAP, recovery phase of MAP; A_MSAP, acute phase of MSAP; R_MSAP, recovery phase of MSAP; A_SAP, acute phase of SAP; R_SAP, recovery phase of SAP. *0.01\u0026lt;p≤0.05, ns: not significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S4 \u003c/strong\u003eDifference analysis of gut microbiota species in the recovery phase of SAP\u003c/p\u003e\n\u003cp\u003eA: Difference analysis of gut microbiota species in SAP vs. CON; B: difference analysis of gut microbiota species in the recovery phase of SAP vs. SAP. A_SAP, acute phase of SAP; R_SAP, recovery phase of SAP. *0.01\u0026lt;p≤0.05.\u003c/p\u003e","description":"","filename":"SupplementaryFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5453055/v1/d6936847449b5dbb61ebe085.pdf"},{"id":71876448,"identity":"c21a3e3c-572b-4652-84ce-88999b6971ec","added_by":"auto","created_at":"2024-12-19 11:00:03","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12761,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S1 \u003c/strong\u003eClinical information of enrolled healthy controls and AP patients.\u003c/p\u003e\n\u003cp\u003eMAP, mild acute pancreatitis; MSAP, moderately severe acute pancreatitis; SAP, severe acute pancreatitis.\u003c/p\u003e","description":"","filename":"TableS1ClinicalinformationofenrolledhealthycontrolsandAPpatients.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5453055/v1/5cb017dd1adb048e4ae0b736.xlsx"},{"id":71876973,"identity":"83bb9947-a2d5-4c10-93df-b847daed7df7","added_by":"auto","created_at":"2024-12-19 11:08:03","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":241134,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S2 \u003c/strong\u003eDifference analysis of gut microbiota species in the recovery phase of AP.\u003c/p\u003e","description":"","filename":"TableS2DifferenceanalysisofgutmicrobiotaspeciesintherecoveryphaseofAP.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5453055/v1/e444656ad026b8e5601b8b14.xlsx"},{"id":71876975,"identity":"0bdc45bf-e8c4-4ba6-b710-91abb81e9f08","added_by":"auto","created_at":"2024-12-19 11:08:03","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":232751,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S3 \u003c/strong\u003eDifference analysis of gut microbiota species in the recovery phase of AP with different severity.\u003c/p\u003e","description":"","filename":"TableS3DifferenceanalysisofgutmicrobiotaspeciesintherecoveryphaseofAPwithdifferentseverity.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5453055/v1/544e1848457f3d0cb2dc8ca3.xlsx"},{"id":71876454,"identity":"c30d41a0-b79c-4c03-8880-1d31284b0711","added_by":"auto","created_at":"2024-12-19 11:00:03","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":116460,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S4\u003c/strong\u003e KEGG functional composition difference analysis of the microbiota in the recovery phase of AP.\u003c/p\u003e","description":"","filename":"TableS4KEGGfunctionalcompositiondifferenceanalysisofthemicrobiotaintherecoveryphaseofAP.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5453055/v1/72bf254fca08637cc0911532.xlsx"},{"id":71878827,"identity":"d49a4ae8-32d9-490a-a8f8-c0aecadb400c","added_by":"auto","created_at":"2024-12-19 11:16:03","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":168094,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S5\u003c/strong\u003e KEGG functional composition difference analysis of the microbiota in the recovery phase of AP with different severity.\u003c/p\u003e","description":"","filename":"TableS5KEGGfunctionalcompositiondifferenceanalysisofthemicrobiotaintherecoveryphaseofAPwithdifferentseverity.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5453055/v1/2498a551688dac37dad680d6.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Metagenomics reveals functional profiles of gut microbiota during the recovery phase of acute pancreatitis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAcute pancreatitis (AP) is a common life-threatening disease of the pancreas. Non-mild AP has an acute onset, rapid progression, and poor prognosis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The course of AP often lasts from tens of days to months, depending on disease severity. Based on its clinical characteristics, AP can generally be divided into three phases: acute reaction, progression, and recovery. While not all patients experience a three-phase course, every surviving patient undergoes a recovery phase [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePancreatic regeneration and functional repair may occur during the acute reaction period. When inflammation occurs, organ repair and regeneration mechanisms are activated, which continue throughout the course of the disease. During the acute reaction and progression phases, the pathophysiological processes are the injury and necrosis of pancreatic cells as well as inflammatory responses. After entering the recovery period, pancreatic regeneration and functional repair become the primary pathophysiological processes with inflammatory responses gradually weakening or attaining a balance [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. At this stage, relief of discomfort, regression of inflammation, and recovery of gastrointestinal function occur as responses to treatment and enhance the quality of life of patients [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, a risk of deterioration exists.\u003c/p\u003e \u003cp\u003eThe human intestinal barrier system consists of the gut flora, the mucosal immune system, and the mucosal epithelium. The stability of gut flora depends on intestinal barrier function [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Inflammatory responses, bacterial translocation, and impaired intestinal barrier are essential for AP progression and complications [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eEnterococcus\u003c/em\u003e, and \u003cem\u003eEnterobacteriaceae\u003c/em\u003e are the primary pathogens that cause secondary infections in AP [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Additionally, dysbiosis of the intestinal flora causes a decrease in the levels of short-chain fatty acid-producing bacteria, which affects the integrity of the intestinal barrier and worsens clinical conditions [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Although previous studies have reported potential routes and mechanisms for the development of AP, more information on intestinal dysfunction over the entire course of AP is required, especially during the recovery phase [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we employed metagenomic sequencing technology to investigate the gut microbiota composition and function in patients with AP of varying severities during the acute and recovery phases, revealing the complex interaction between gut microbes and the host.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003ePatients\u003c/h2\u003e\n \u003cp\u003eFrom January 2021 to September 2021, patients diagnosed based on the 2012 Revision of the Atlanta Classification and admitted to the Peking Union Medical College Hospital, Beijing, China, were included in this study. Patients were enroled within 48 h of disease onset and followed up for 30 d to observe their recovery. Patients were excluded if they had a history of immunodeficiency, inflammatory bowel disease, specificity, asthma, irritable bowel syndrome, celiac disease, gastroenteritis, colon cancer, human immunodeficiency virus infection, necrotising enterocolitis, or arthritis. The criteria for individuals recovering from AP were as follows: (1) relief of abdominal pain in patients with mild AP (MAP); (2) decrease in the improved Marshall score to 0 points in patients with moderately severe AP (MSAP) or severe AP (SAP); and (3) tolerable enteral nutrition. The control group was defined as described in our previous study [10]. Four healthy individuals were recruited and matched for age, sex, and body mass index. Written informed consent was obtained from all the patients. Approval was obtained from the Institutional Review Board of Peking Union Medical College Hospital (No. JS1826).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eSample collection\u003c/h3\u003e\n\u003cp\u003eA flowchart of the study is shown in Figure S1. Rectal swabs were obtained from each patient with AP immediately after admission and during the recovery phase, owing to the difficulties in direct fecal collection due to gastrointestinal dysfunction [14, 15]. The control group consisted of four healthy volunteers, with all individuals having empty stomachs before sample collection. All individuals were enroled at Peking Union Medical College Hospital, Beijing, China. Fecal sample collection methods have been previously described in detail [10]. Soap, water, and 70% alcohol were used to remove the anus. The disinfectant was evaporated independently to reduce commensal skin contamination. The sterile swab was infiltrated with normal saline for 2 min before being inserted into the anus as deep as 4–5 cm and then rotated gently to obtain fecal samples. Rectal swabs with fecal samples were quickly placed into a sterile tube and immediately stored at -80℃ before shipping to the laboratory under cold chain.\u003c/p\u003e\n\u003ch3\u003eMetagenomics sequencing, quality control, and genome assembly\u003c/h3\u003e\n\u003cp\u003eDNA was extracted from the fecal samples according to the manufacturer’s instructions. DNA purity, concentration, and quality were determined using NanoDrop2000 (Thermo Fisher Scientific, Waltham, MA, USA), TBS-380, and a 1% agarose gel electrophoresis system, respectively. Paired-end sequencing was performed on an Illumina HiSeq4000 platform (Illumina, San Diego, CA, USA). After removing the low-quality reads, the reads were aligned to the human genome using BWA (v 0.7.9a) [16]. After quality control, the reads were mapped to representative genes with 95% identity using SOAPaligner (v 2.21, http://soap.genomics.org.cn/) [17], and gene abundance was assessed for each sample.\u003c/p\u003e\n\u003ch3\u003eAlpha diversity and principal component analysis\u003c/h3\u003e\n\u003cp\u003eα-diversity analysis was performed using the R packages “vegan” (v 4.0.5) and “reshape2” (v 4.0.5). Species diversity was assessed using Shannon’s index, with a higher value indicating higher floral diversity. In addition, principal component analysis, which reflects the differences and distances between samples by analysing the community composition of different samples, was performed. The relatively similar mean values of the samples from different groups indicated a similarity in species composition.\u003c/p\u003e\n\u003ch3\u003eAnalysis of typing and dominant microbiome composition\u003c/h3\u003e\n\u003cp\u003eMicrobiome typing analysis involves typing of the dominant microflora structures in different samples using statistical clustering. Through this method, different samples with structures similar to those of the dominant microbiome can be grouped into one class. This method is mainly applied to the specific environmental samples. Based on the relative abundance of the microbiome at the genus level, the Bray–Curtis distance was calculated for clustering. After calculating the optimal clustering K using the Calinski–Harabasz index, the 28 samples were divided into different types. In patients with AP and those recovering from AP, a Circos diagram was used to visualise the distribution of the dominant microflora among different samples.\u003c/p\u003e\n\u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003eDifferential microbiome analysis and functional annotation\u003c/h2\u003e\n \u003cp\u003eBased on the read abundance data, species with significant differences were identified at P \u0026lt; 0.05, using the Wilcoxon rank-sum test. Nonredundant genes were annotated against the KEGG database (v 94.2, http://www.genome.jp/keeg/) [18] using Diamond (v 2.0.13, http://ab.inf.uni-tuebingen.de/software/diamond/) [19] at an optimised e-value (cutoff of 1e − 5).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eStatistical analyses\u003c/h3\u003e\n\u003cp\u003eStatistical analyses and data visualisation were conducted using custom scripts that are publicly available (https://cloud.majorbio.com/page/project/task.html?project_id=7df904c5bhnc1vgo2oj2fkevfr) [20]. The Strengthening The Organizing and Reporting of Microbiome Studies checklist was completed and uploaded to Zenodo (https://zenodo.org/records/10676912?token=eyJhbGciOiJIUzUxMiJ9.eyJpZCI6ImM1YTIzYmI0LTU0MmYtNDQ4NS05NzdkLTVmOTRkOWFmNGI0NCIsImRhdGEiOnt9LCJy\u003cbr/\u003eYW5kb20iOiIxNjE4NmI1NWVmNjc1ZTY4YjYyMjcxOTRkNTI5NzRlMyJ9.T3S5fNFyjdfxPgK-BGoCYPaVx4H7gKLMLGcJ8cYuogIp9mIhJkdl1QUqH9vJm5qaEeZmEmue2Jqqc1XCFLo28A).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePatient characteristics\u003c/h2\u003e \u003cp\u003eTwelve patients with AP and four healthy controls were included in this study. Among the 12 patients with AP, 4 had MAP, 5 had MSAP, and 3 had SAP. After treatment, the clinical condition of all patients with AP initially improved and their serum lipase and amylase levels recovered or met the expected values. Meanwhile, APACHE II and Balthazar CT scores decreased during the recovery phase. Detailed information on the patients with AP and healthy controls is presented in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eGut microbiome diversity in patients with AP during acute and recovery phases\u003c/h2\u003e \u003cp\u003eCompared with that in healthy controls, the α-diversity (Shannon index) in patients with AP was significantly reduced during the acute phase and further decreased during the recovery phase, despite improvement of clinical manifestations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). A reduction in diversity was observed among different severities during the recovery phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). During the recovery period, clinical manifestations started to improve but gut microbiota diversity did not recover concomitantly. Thus, the recovery of gut microbiota and intestinal function was slower than the overall clinical improvement. Category-based principal coordinate analysis (PCoA) revealed differences in β-diversity between the control and AP groups, showing distinct clustering. However, no significant clustering was observed between the acute and recovery phases or among the MAP, MSAP, and SAP subgroups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The structural diversity of acute-phase samples only slightly differed at different severity levels (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). During the recovery phase, the samples with different severities exhibited clear clustering.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of enterotypes and dominant microbiome composition during the recovery phase of AP\u003c/h2\u003e \u003cp\u003eThe 28 samples were clustered into two distinct enterotypes, with those having a high abundance of the \u003cem\u003eBacteroides\u003c/em\u003e genus being classified as enterotype 1 and those having a high abundance of the \u003cem\u003eEnterococcus\u003c/em\u003e genus classified as enterotype 2 for the total group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and subgroups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). \u003cem\u003eEnterococcus\u003c/em\u003e was the dominant genus in the gut microbiota during the acute and recovery phases of AP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). At the species level, the abundances of \u003cem\u003eunclassified_g__Enterococcus\u003c/em\u003e and \u003cem\u003eEnterococcus_faecium\u003c/em\u003e were high during the acute and recovery phases of AP, which is in accordance with the results at the genus level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Similarly, in this subgroup, \u003cem\u003eEnterococcus\u003c/em\u003e was the dominant genus in the gut microbiota during the acute and recovery phases of AP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). At the species level, the abundances of \u003cem\u003eunclassified_g__Enterococcus\u003c/em\u003e and \u003cem\u003eEnterococcus_faecium\u003c/em\u003e were high during the acute and recovery phases of AP, which is in accordance with the results at the genus level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Comparison of the acute and recovery phases of MAP revealed a decrease in the abundance of \u003cem\u003eEnterococcus\u003c/em\u003e (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA) and an increase in that of \u003cem\u003eBacteroidales\u003c/em\u003e (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB) increased (without significant differences). Conversely, comparison of the acute and recovery phases of MSAP revealed an increase in the abundance of \u003cem\u003eEnterococcus\u003c/em\u003e (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eC) and a decrease in that of \u003cem\u003eBacteroidales\u003c/em\u003e (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eD) (without significant differences). Similarly, comparison of the acute and recovery phases of SAP revealed an increase in the abundance of \u003cem\u003eEnterococcus\u003c/em\u003e (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eE) and a decrease in that of \u003cem\u003eBacteroidales\u003c/em\u003e (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eF) (without significant differences). Thus, \u003cem\u003eEnterococcus\u003c/em\u003e was the dominant genus of the gut microbiota during recovery.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDifferential microbiome analysis during the recovery phase of AP\u003c/h2\u003e \u003cp\u003eDifferential species composition analysis showed that \u003cem\u003eunclassified_g__Enterococcus\u003c/em\u003e was the most abundant species during the acute phase of AP (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). During the recovery phase, the abundance of beneficial bacteria such as \u003cem\u003eFaecalibacterium_prausnitzii\u003c/em\u003e, \u003cem\u003eBifidobacterium_longum\u003c/em\u003e, and \u003cem\u003eFirmicutes_bacterium\u003c/em\u003e was low, indicating that the overall abundance of beneficial bacteria had not recovered during this period (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). At the second level (90 species of bacteria in the acute and recovery phases of AP) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), 29 species showed an inverse trend in abundance during the acute and recovery phases in patients with AP (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The short-chain fatty acid-producing bacteria \u003cem\u003eAnaerococcus\u003c/em\u003e, \u003cem\u003eBlautia\u003c/em\u003e, \u003cem\u003eCandidatus_Blautia_pullistercoris\u003c/em\u003e, and \u003cem\u003eEubacterium_sp._CAG:86\u003c/em\u003e were low in abundance during the recovery phase. The abundance of the propionic and butyric acid-producing bacterium \u003cem\u003eRoseburia_inulinivorans_CAG:15\u003c/em\u003e was low during the acute phase of AP, whereas that of \u003cem\u003eCandidatus_Eisenbergiella_pullicola\u003c/em\u003e was high. The levels of anti-inflammatory microbiota began to increase during the recovery phase; however, those of other beneficial bacteria did not return to normal.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe 29 kinds of bacteria showed a reverse trend in abundance levels between the acute and recovery phases of AP patients\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAcute phase vs Control\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRecovery phase vs Acute phase\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__unclassified_g__Anaerococcus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Beta_vulgaris\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Alistipes_senegalensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Paludibacteraceae_bacterium\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Blautia_sp._CAG:52\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Cajanus_cajan\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Roseburia_inulinivorans_CAG:15\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Pelagicola_marinus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Corynebacterium_urealyticum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Pseudoramibacter_alactolyticus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Trifolium_pratense\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Helianthus_annuus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Dorea_longicatena_CAG:42\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Oryza_sativa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Dorcoceras_hygrometricum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Candidatus_Blautia_pullistercoris\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Juglans_regia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Candidatus_Eisenbergiella_pullicola\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Rosa_chinensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Hevea_brasiliensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__bacterium_1XD8-92\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Diploscapter_pachys\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Fallopia_multiflora\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Eubacterium_sp._CAG:86\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Corynebacterium_massiliense\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Panicum_virgatum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Clostridium_sp._CCUG_7971\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Pseudoglutamicibacter_albus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Methanobrevibacter_oralis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eComparison of the number of species between patients with MAP and healthy controls revealed 498 significantly different species of gut microbiota. The levels of \u003cem\u003eEnterococcus\u003c/em\u003e and \u003cem\u003eBacteroidales\u003c/em\u003e significantly increased and decreased, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Comparison of the acute and recovery phases of MAP revealed 13 significantly different species of gut microbiota. The abundances of \u003cem\u003eOscillibacter\u003c/em\u003e, \u003cem\u003eRuminococcus\u003c/em\u003e, and \u003cem\u003eFirmicutes\u003c/em\u003e significantly decreased during the recovery phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Comparison of patients with MSAP and healthy controls revealed 1223 significantly different species of gut microbiota. \u003cem\u003eEnterococcus\u003c/em\u003e was the most significantly increased genus in the gut microbiota (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In the MSAP group, 241 significantly different species were identified between the recovery and acute phases. \u003cem\u003eAdlercreutzia equolifaciens\u003c/em\u003e showed the most significant decrease in abundance; however, its abundance was low during the recovery phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Comparison of patients with SAP and healthy controls revealed 397 significantly different species of gut microbiota. The abundances of \u003cem\u003eEnterococcus\u003c/em\u003e and \u003cem\u003ePorphyromonas asaccharolytica\u003c/em\u003e significantly increased and decreased, respectively (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA). No significant differences in gut microbiota species between the acute and recovery phases of SAP were found (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eB). Our results suggest that patients with SAP may have recovered from their clinical symptoms; however, their microbiota profiles have not yet recovered. \u003cem\u003eEnterococcus\u003c/em\u003e was the most abundant genus in the acute phase of AP at varying severities.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTwo common species were identified between the 498 differential species during the acute phase of MAP and 13 differential species during the recovery phase of MAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). \u003cem\u003eFirmicutes_bacterium_CAG:129_59_24\u003c/em\u003e, belonging to Firmicutes, showed low abundance during the recovery phase of MAP, indicating that its function had not yet recovered. \u003cem\u003eIntestinimonas_timonensis\u003c/em\u003e, a pro-inflammatory microorganism, showed decreased abundance during the recovery phase of MAP, indicating that the inflammatory function of the species gradually recovered. Between the 1223 differential species during the acute phase of MSAP and 241 differential species during the recovery phase, 47 common species were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Of these, 19 were highly abundant in patients with MSAP during the acute phase but showed low abundance during the recovery phase (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Among them, \u003cem\u003eCandidatus_Eisenbergiella_pullicola\u003c/em\u003e and \u003cem\u003eCollinsella_provencensis\u003c/em\u003e, which are related to intestinal wall permeability, \u003cem\u003eOlsenella_sp._Marseille-P4518\u003c/em\u003e, which is a fructose-consuming strain, \u003cem\u003eClostridium_sp._D53t1_180928_C8\u003c/em\u003e, \u003cem\u003eBlautia_sp._AM28-10\u003c/em\u003e, and \u003cem\u003eOlsenella_sp._Marseille-P4518\u003c/em\u003e were highly abundant during the acute phase of MSAP but decreased in abundance during the recovery phase.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe 19 kinds of bacteria showed a reverse trend in abundance levels between the acute and recovery phase of MASP patients\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA_MSAP vs Control\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR_MSAP vs A_MSAP\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Candidatus_Eisenbergiella_pullicola\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Prunus_dulcis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Hevea_brasiliensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Myoviridae_sp._ct3Oc10\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Arachis_hypogaea\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Fallopia_multiflora\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Trema_orientale\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Collinsella_provencensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Candidatus_Aphodovivens_avistercoris\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Clostridium_sp._CCUG_7971\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Candidatus_Blautia_pullistercoris\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Olsenella_sp._Marseille-P4518\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Pogostemon_cablin\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Betaproteobacteria_bacterium\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Clostridium_sp._D43t1_170807_H7\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Blautia_sp._AM28-10\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Turicibacter_sp._H121\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Methanobrevibacter_oralis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003es__Clostridium_sp._D53t1_180928_C8\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edown\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eA_MSAP, acute phase of MSAP; R_MSAP, recovery phase of MSAP.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of differences in functional compositions in the recovery phase of AP\u003c/h2\u003e \u003cp\u003eThe gut microbiota of patients with acute-phase AP was involved in 87 significantly different functional compositions of the KEGG pathway. Adenosine triphosphate-binding cassette (ABC) transporters, fructose and mannose metabolism, and activation of the phosphotransferase system (PTS) were significantly upregulated, whereas glycine, serine, and threonine metabolism was significantly downregulated during the acute phase of AP (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Meanwhile, 13 significantly different functional pathways were identified during the recovery phase, of which microbial metabolism was significantly activated in diverse environments. However, RNA degradation, protein processing in the endoplasmic reticulum, ribosome biogenesis in eukaryotes, and other RNA- and protein-related pathways were inhibited or downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Only three pathways were shared between acute and recovery phases: RNA degradation, flavonoid biosynthesis, and stilbenoid, diarylheptanoid, and gingerol biosynthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). The changes in pathway function for \\ patients with varying AP severities are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003e and summarised in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. We identified 37, 98, and 23 significantly different KEGG functional compositions in patients with MAP, MSAP, and SAP, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eKEGG functional composition difference analysis of the microbiota in the recovery phase of AP with different severity.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAcute phase\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRecovery phase\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMAP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhosphotransferase System (PTS) \u0026uarr;\u003c/p\u003e \u003cp\u003eGlycerolipid Metabolism \u0026uarr;\u003c/p\u003e \u003cp\u003eGlycine, serine, and threonine metabolism \u0026darr;\u003c/p\u003e \u003cp\u003eGlyoxylate and dicarboxylate metabolism \u0026darr;\u003c/p\u003e \u003cp\u003eCitrate cycle (TCA cycle) \u0026darr;\u003c/p\u003e \u003cp\u003eButanoate metabolism \u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCardiac muscle contraction \u0026darr;\u003c/p\u003e \u003cp\u003emTOR signaling pathway \u0026darr;\u003c/p\u003e \u003cp\u003ePhagosome \u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMSAP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eABC transporters \u0026uarr;\u003c/p\u003e \u003cp\u003ePhosphotransferase system (PTS) \u0026uarr;\u003c/p\u003e \u003cp\u003eFructose and mannose metabolism \u0026uarr;\u003c/p\u003e \u003cp\u003eCarbon metabolism \u0026darr;\u003c/p\u003e \u003cp\u003eAmino sugar and nucleotide sugar metabolism \u0026darr;\u003c/p\u003e \u003cp\u003eGlycine, serine, and threonine metabolism \u0026darr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSAP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNucleocytoplasmic transport \u0026uarr;\u003c/p\u003e \u003cp\u003eSteroid biosynthesis \u0026uarr;\u003c/p\u003e \u003cp\u003eInflammatory mediator regulation of TRP channels \u0026uarr;\u003c/p\u003e \u003cp\u003eViral myocarditis \u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe onset, development, and recovery of AP are closely related to intestinal flora dysbiosis and bacterial translocation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The intestinal flora can participate in the onset and recovery of AP by influencing host metabolism and intestinal mucosal permeability [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. We performed shotgun metagenomic sequencing on 12 patients with AP during the acute and recovery phases. Significant microbiome dysbiosis was observed during both phases, suggesting an association between dysbiosis and recovery from AP.\u003c/p\u003e \u003cp\u003e \u003cem\u003eEnterococcus\u003c/em\u003e, a harmful bacterium, was the most abundant genus present during the acute and recovery phases of AP. For example, the relative abundances of \u003cem\u003es__Enterococcus_faecium\u003c/em\u003e, \u003cem\u003es__Enterococcus_faecalis\u003c/em\u003e, \u003cem\u003es__Enterococcus_hirae\u003c/em\u003e, \u003cem\u003es__Enterococcus_gallinarum\u003c/em\u003e, and \u003cem\u003es__Enterococcus_casseliflavus\u003c/em\u003e were high during the acute phase of AP. \u003cem\u003eEnterococcus\u003c/em\u003e can attach to and penetrate host cells, traverse host epithelial barriers, and gain entry into the systemic circulation and other organs, promoting its spread within the host [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The relative abundance of \u003cem\u003eEnterococcus\u003c/em\u003e spp. is associated with infection and systemic inflammation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Biofilm formation by \u003cem\u003eEnterococcus\u003c/em\u003e involves a complex interplay between genes and virulence factors, including gelatinases, cytolysins, secretory antigen A, fimbriae, microbial surface components that recognise adhesion matrix molecules, and DNA [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Zhang et al. reported that the relative abundance of the potentially pathogenic genus \u003cem\u003eEnterococcus\u003c/em\u003e increased in the SAP group but decreased after Qingyi decoction treatment [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. As a 0000000, \u003cem\u003eEnterococcus faecium\u003c/em\u003e performed well in distinguishing patients with acute necrotising pancreatitis from those with non-necrotising pancreatitis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In patients with Crohn's disease, ulcerative colitis, or ischemic colitis, the abundance of potentially pathogenic bacteria, such as \u003cem\u003eEnterococcus faecium\u003c/em\u003e and \u003cem\u003eEnterococcus faecalis\u003c/em\u003e, is increased, indicating their potential diagnostic value [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDuring the recovery phase of AP, the abundances of the beneficial bacteria \u003cem\u003eFaecalibacterium_prausnitzii\u003c/em\u003e, \u003cem\u003eBifidobacterium_longum\u003c/em\u003e, and \u003cem\u003eFirmicutes_bacterium\u003c/em\u003e were significantly lower than those during the acute phase. These results indicate that the clinical manifestations began to improve, but the overall abundance of beneficial bacteria did not recover concomitantly during the AP recovery period. Recovery of the gut microbiota and intestinal function was slower than the overall clinical improvement. \u003cem\u003eFaecalibacterium_prausnitzii\u003c/em\u003e is involved in inflammatory processes, blocking nuclear factor kappa B activation and interleukin (IL)-8 production [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The abundance of \u003cem\u003eFaecalibacterium prausnitzii\u003c/em\u003e was lower in patients with chronic pancreatitis (CP) and diabetes than in those without diabetes and in controls [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Similarly, McEachron et al.. observed a significantly decreased abundance of \u003cem\u003eFaecalibacterium\u003c/em\u003e in patients with CP [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. \u003cem\u003eBifidobacterium\u003c/em\u003e has been reported to reduce the risk of multiple organ failure in SAP [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. \u003cem\u003eBifidobacteria\u003c/em\u003e, with reduced abundance in feces, is inversely correlated with the severity of the systemic inflammatory response in patients with AP [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. \u003cem\u003eFirmicutes\u003c/em\u003e, which are pathogenic Gram-positive bacteria, are mainly butyrate producers and have been identified in patients with AP [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. \u003cem\u003eAnaerococcus\u003c/em\u003e, \u003cem\u003eBlautia\u003c/em\u003e, \u003cem\u003eCandidatus_Blautia_pullistercoris\u003c/em\u003e, \u003cem\u003eEubacterium_sp._CAG:86\u003c/em\u003e, and other short-chain fatty acid-producing bacteria were significantly lower in abundance during the recovery phase of AP. In our previous study, we observed a significantly increased abundance of \u003cem\u003eAnaerococcus\u003c/em\u003e in MSAP and a significantly decreased abundance of \u003cem\u003eBlautia\u003c/em\u003e in MAP [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The abundance of the propionic and butyric acid-producing bacterium \u003cem\u003eRoseburia_inulinivorans_CAG:15\u003c/em\u003e was significantly lower during the recovery phase, whereas that of \u003cem\u003eCandidatus_Eisenbergiella_pullicola\u003c/em\u003e was significantly higher during the acute phase of AP. \u003cem\u003eFaecalibacterium\u003c/em\u003e and \u003cem\u003eRoseburia\u003c/em\u003e are well-known butyrate producers that attenuate intestinal inflammation and are beneficial to the host [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The relative abundance of \u003cem\u003eRoseburia\u003c/em\u003e decreased in mice with CP [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. \u003cem\u003eEisenbergiella\u003c/em\u003e has been suggested to protect mice with ulcerative colitis by reducing the intestinal inflammatory response [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Bacterial abundance decreased significantly during the recovery phase, indicating an improvement in inflammation-related microbiota function. However, the abundance of beneficial bacteria did not fully recover during the AP recovery phase.\u003c/p\u003e \u003cp\u003eIn MAP, the abundance of \u003cem\u003eOscillibacter_sp.\u003c/em\u003e significantly increased during the acute phase but decreased during the recovery phase. Increased abundance of \u003cem\u003eOscillibacter\u003c/em\u003e is associated with intestinal permeability and host inflammation in patients with large-artery atherosclerotic stroke or transient ischemic attacks [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. \u003cem\u003eOscillibacter\u003c/em\u003e sp. 57_20 has been reported to identify SAP at an early stage [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In MSAP, the abundances of \u003cem\u003eClostridium_sp._D53t1_180928_C8\u003c/em\u003e and \u003cem\u003eBlautia_sp._AM28-10\u003c/em\u003e significantly increased during the acute phase but decreased during the recovery phase. \u003cem\u003eClostridium perfringens\u003c/em\u003e was isolated from cultures of pancreatic tissue obtained from a patient with AP during hospitalisation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Significant differences in \u003cem\u003eCollinsella\u003c/em\u003e abundance were observed between the MAP and SAP groups [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The abundance of \u003cem\u003eCollinsella\u003c/em\u003e is associated with the production of the proinflammatory cytokine IL-17A [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Decreased abundance of \u003cem\u003eCollinsella\u003c/em\u003e has been reported in children with CP [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In addition, \u003cem\u003eCandidatus_Eisenbergiella_pullicola\u003c/em\u003e and \u003cem\u003eCollinsella_provencensis\u003c/em\u003e were associated with intestinal wall permeability. In SAP, the abundance of \u003cem\u003eAdlercreutzia_equolifaciens\u003c/em\u003e was high during the acute phase but decreased during recovery. However, no significant difference in its abundance was found between the two phases, suggesting a possible intrinsic role for SAP.\u003c/p\u003e \u003cp\u003eAdditionally, we observed that disturbances in gut microbiota affect host metabolism and molecular transport mechanisms. PTS was significantly activated, whereas glycine, serine, and threonine metabolism was significantly inhibited during the acute phase of AP. PTS, one of the most efficient sugar transport systems, plays a crucial role in the regulation of glucose metabolism in bacteria [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Compared with their levels 6 weeks after hospital discharge, the levels of glycine, serine, and threonine in patients with AP were significantly lower upon admission [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Glycine administration improves AP pathomorphology and reduces the severity of MAP and SAP [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. During the AP recovery phase, microbial metabolism is significantly activated and upregulated in diverse environmental pathways. However, RNA degradation, protein processing in the endoplasmic reticulum, ribosome biogenesis in eukaryotes, and other functional pathways related to RNA and proteins were inhibited and downregulated. These findings indicate that while intestinal function and the gut microbiota structure start to recover, the restoration of RNA- and protein-related biological processes lags behind.\u003c/p\u003e \u003cp\u003eDuring the recovery phase of MAP, an increase in the functional composition of glycine, serine, and threonine metabolism and a decrease in the functional composition of the PTS compared with the acute phase was observed. In addition, cardiac muscle contraction was the most significantly reduced functional component during the MAP recovery phase. The PTS regulates carbohydrate uptake and is considered a potential biomarker of inflammatory bowel disease [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Glycine, serine, and threonine metabolism is involved in glycolytic biosynthesis in pancreatic cancer [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Regardless of its severity, AP is accompanied by cardiac injury, and the degree of damage is associated with the clinical type of AP [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Thus, activation of the glycine, serine, and threonine metabolic pathways, deactivation of the phosphotransferase system, and reduction of cardiac muscle contraction are associated with the recovery phase in patients with MAP. Moreover, during the recovery phase of MSAP, an increase in the functional composition of carbon metabolism and a decrease in the functional composition of ABC transporters compared with the acute phase was observed. ABC transporters translocate various endogenous metabolites and cytotoxic compounds across the lipid bilayer [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In cancer, overexpression of ABC transporters leads to drug resistance [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In mice with AP, antioxidant treatment with carbon monoxide-bound hemoglobin vesicles effectively reduced the inflammatory response and pancreatic tissue damage [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Therefore, carbon metabolism and ABC transporters may be involved in MSAP recovery. These signalling pathways exhibit an opposite trend during the recovery phase of MAP and MSAP in comparison with the acute phase, suggesting that they may benefit recovery.\u003c/p\u003e \u003cp\u003eHowever, this study had several limitations. The sample size used in shotgun metagenomics was relatively small, and further studies with more participants are warranted. In addition, the causal links among the microbiota, functional composition, and AP recovery require further investigation.\u003c/p\u003e \u003cp\u003eIn conclusion, we identified the presence of several bacteria, along with their functional composition, during the recovery phase of AP through a shotgun metagenomic survey, extending the current understanding of gut microbiota during this phase. This study provides information that may guide the development of treatment strategies and the prognosis of AP.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAP, Acute pancreatitis\u003c/p\u003e\n\u003cp\u003eMAP, mild AP\u003c/p\u003e\n\u003cp\u003eMSAP, moderately severe AP\u003c/p\u003e\n\u003cp\u003eSAP, severe AP\u003c/p\u003e\n\u003cp\u003ePCoA, principal coordinate analysis\u003c/p\u003e\n\u003cp\u003eABC,\u0026nbsp;Adenosine triphosphate-binding cassette\u003c/p\u003e\n\u003cp\u003ePTS, phosphotransferase system\u003c/p\u003e\n\u003cp\u003eCP, chronic pancreatitis\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional review board of the Peking Union Medical College Hospital (No. JS1826) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study. Informed consent was obtained from all individual participants included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData availability\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data had been deposited in the NCBI Sequence Read Archive (BioProject accession ID: PRJNA1074432, http://www.ncbi.nlm.nih.gov/bioproject/1074432). The full results of all analyses were included in Table S2-5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the National Natural Science Foundation of China (grant number 32170788), the National High Level Hospital Clinical Research Funding (grant number 2022-PUMCH-B-023), the National Key Clinical Specialty Construction Project (grant number ZK108000), and Natural Science Foundation of Beijing (grant number 7232123).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgments\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extend our sincere gratitude to Taylor \u0026amp; Francis Editing Services for their meticulous editing and valuable contributions in enhancing the quality and clarity of our manuscript.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eX.S., J.N.L., D.W.* and Q.W.: study design, interpretation of data, and drafting of the manuscript. X.S., J.N.L., and D.W.\u003csup\u003e\u0026dagger;\u003c/sup\u003e contributed equally to this work and are co-first authors. Z.Y.H., X.X.Y., and Z.H.Y.: sample collection, statistical analysis, and critical revision of the manuscript. J.X., Q.W. and D.W.#: study concept and critical revision. D.W.#: final approval of the manuscript. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGukovskaya, A. S. et al. Recent Insights Into the Pathogenic Mechanism of Pancreatitis: Role of Acinar Cell Organelle Disorders. \u003cem\u003ePancreas\u003c/em\u003e. \u003cstrong\u003e48,\u003c/strong\u003e 459-470 (2019).\u003c/li\u003e\n\u003cli\u003eHammer, H. F. 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Carbon monoxide-bound hemoglobin vesicles ameliorate multiorgan injuries induced by severe acute pancreatitis in mice by their anti-inflammatory and antioxidant properties. \u003cem\u003eInt J Nanomedicine\u003c/em\u003e. \u003cstrong\u003e11,\u003c/strong\u003e 5611-5620 (2016).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"acute pancreatitis, recovery phase, shotgun metagenomics, gut microbiota, microbiota composition dysbiosis","lastPublishedDoi":"10.21203/rs.3.rs-5453055/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5453055/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGut microbiota play a critical pathogenic role in acute pancreatitis (AP). This study aimed to investiage the gut microbiota composition and function during the recovery phase of AP. Rectal swab samples obtained from 12 AP patients of varying severity in the acute and recovery phases were sequenced using shotgun metagenomic sequencing. The α-diversity, principal components, typing, and dominant microbiome composition were analyzed, followed by a difference analysis of gut microbiota composition and functional enrichment. In the recovery phase of AP, the microbial diversity remained decreased, and there were minimal difference in the structural diversity of the microbiome. There was an increasing tendency of beneficial bacteria (Bacteroidales) and a decreasing tendency of harmful bacteria (Enterococcus and Firmicutes) in the recovery phase of mild AP (MAP). However, in the recovery phase of moderately severe AP (MSAP) and severe AP, Enterococcus abundance increased compared with that in the acute phase. Some signaling pathways changed in the opposite direction in the recovery phase of MAP and MSAP compared to the acute phase. These results suggested that gut microbiome composition and function are associated with AP recovery, which may be used to develop strategies for the treatment and prognosis of AP.\u003c/p\u003e","manuscriptTitle":"Metagenomics reveals functional profiles of gut microbiota during the recovery phase of acute pancreatitis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-19 10:59:57","doi":"10.21203/rs.3.rs-5453055/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-01-13T07:49:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-11T23:02:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-24T15:42:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"251415144214873161450035251565191956372","date":"2024-12-22T15:19:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"73287985153192059320891125743836404070","date":"2024-12-20T22:56:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-12-20T12:30:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-12-12T12:16:17+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-11-18T11:12:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-18T04:17:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-11-14T09:57:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4ed10b5e-b6e2-488e-b8e8-d58ff4ca4731","owner":[],"postedDate":"December 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":41564236,"name":"Biological sciences/Microbiology/Communities"},{"id":41564237,"name":"Health sciences/Gastroenterology/Gastrointestinal diseases/Pancreatic disease/Pancreatitis"}],"tags":[],"updatedAt":"2025-07-07T15:59:34+00:00","versionOfRecord":{"articleIdentity":"rs-5453055","link":"https://doi.org/10.1038/s41598-025-05127-5","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-01 15:56:54","publishedOnDateReadable":"July 1st, 2025"},"versionCreatedAt":"2024-12-19 10:59:57","video":"","vorDoi":"10.1038/s41598-025-05127-5","vorDoiUrl":"https://doi.org/10.1038/s41598-025-05127-5","workflowStages":[]},"version":"v1","identity":"rs-5453055","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5453055","identity":"rs-5453055","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}