Reference gene catalog and metagenome-assembled genomes from the gut microbiome reveal the microbial composition, antibiotic resistome, and adaptability of a lignocellulose diet in the giant panda.

The giant panda, a strict herbivore that feeds on bamboo, still retains a typical carnivorous digestive system. Reference catalogs of microbial genes and genomes are lacking, largely limiting the antibiotic resistome and functional exploration of the giant panda gut microbiome. Here, we integrated 177 fecal metagenomes of captive and wild giant pandas to construct a giant panda integrated gene catalog (GPIGC) comprised of approximately 4.5 million non-redundant genes and reconstruct 393 metagenome-assembled genomes (MAGs). Taxonomic and functional characterization of genes revealed that the captivity of the giant panda significantly changed the core microbial composition and the distribution of microbial genes. Higher abundance and prevalence of antibiotic resistance genes (ARGs) were detected in the guts of captive giant pandas, and ARG distribution was influenced by geography, for both captive and wild individuals. Escherichia, as the prevalent genus in the guts of captive giant pandas, was the main carrier of ARGs, meaning there is a high risk of ARG transmission by Escherichia. We also found that multiple mcr gene variants, conferring plasmid-mediated mobile colistin resistance, were widespread in the guts of captive and wild giant pandas. There were low proportions of carbohydrate-active enzyme (CAZyme) genes in GPIGC and MAGs compared with several omnivorous and herbivorous mammals. Many members of Clostridium MAGs were significantly enriched in the guts of adult, old and wild giant pandas. The genomes of isolates and MAGs of Clostridiaceae harbored key genes or enzymes in complete pathways for degrading lignocellulose and producing short-chain fatty acids (SCFAs), indicating the potential of these bacteria to utilize the low-nutrient bamboo diet. Overall, our data presented an exhaustive reference gene catalog and MAGs in giant panda gut and provided a comprehensive understanding of the antibiotic resistome and microbial adaptability for a high-lignocellulose diet.

Factors influencing bamboo intake of captive giant pandas (Ailuropoda melanoleuca).

Bamboo is the main food source of the giant panda. To increase bamboo intake in captive giant pandas, we studied factors affecting the bamboo intake. Fourteen healthy captive giant pandas in Dujiangyan Base of China Conservation and Research Center for The Giant Panda ("Dujiangyan Base" for short) were selected as research objects. A bamboo feeding experiment was conducted to study the effects of seasons, bamboo age, slope orientations where bamboo grows and felling-feeding time on bamboo intake of the giant panda. We found that the type of bamboo that captive giant pandas feed on was abundant in spring and summer, but relatively homogeneous in winter. With the increase of bamboo age, the intake of bamboo leaves decreased, while bamboo culms increased. The feed intake of 1-year-old bamboo leaves and 5-year-old bamboo culms reached the highest respectively. The slope orientation also affected the panda's bamboo intake, and the bamboo growing on sunny slopes or semi-sunny slopes was more favored by captive giant pandas. Moreover, the bamboo intake reached the highest when felling-feeding time was less than 24 h. In short, we confirmed that seasons, bamboo age, slope orientations and felling-feeding time were factors affecting bamboo intake for captive giant pandas. This study was expected to provide scientific guidance improving the feeding behavior management of captive giant pandas.

Organic Fertilizers Shape Soil Microbial Communities and Increase Soil Amino Acid Metabolites Content in a Blueberry Orchard.

The decline in soil nutrients is becoming a major concern of soil degradation. The possibility of using organic waste as a soil additive to increase nutrients and essential components is significant in soil quality protection and waste management. The aim of this study was to investigate the effects of composted spent mushroom substrate (MS), giant panda feces (PF), and cattle manure (CM) as organic fertilizers in soil microbial communities and metabolites in blueberry orchard in China, which were measured by using high-throughput sequencing and gas chromatography-mass spectrometry (GC-MS)-based metabolomics. Altogether, 45.66% of the bacterial operational taxonomic units (OTUs) and 9.08% of the fungal OTUs were detected in all treatments. Principal coordinates analysis demonstrated that the bacterial and fungal communities in MS and PF treatments were similar, whereas the communities in the not-organic fertilized control (CK) were significantly different from those in the organic fertilizer treatments. Proteobacteria, Acidobacteria, and Bacteroidetes were the dominant bacterial phyla, and Basidiomycota, Ascomycota, and Mortierellomycota the dominant fungal phyla. Redundancy analysis indicated that pH and available potassium were the main factors determining the composition of microbial communities. The fungal genera Postia, Cephalotrichum, and Thermomyces increased in organic fertilizer treatments, and likely promoted the degradation of organic fertilizers into low molecular-weight metabolites (e.g., amino acids). PCA and PLS-DA models showed that the metabolites in CK were different from those in the other three treatments, and those in CM were clearly different from those in MS and PF. Co-occurrence network analysis showed that several taxa correlated positively with amino acid contents. The results of this study provide new insights into organic waste reutilization and new directions for further studies.

Antimicrobial resistance of Escherichia coli, Enterobacter spp., Klebsiella pneumoniae and Enterococcus spp. isolated from the feces of giant panda.

BACKGROUND: Escherichia coli, Enterobacter spp., Klebsiella pneumoniae and Enterococcus spp., common gut bacteria in giant pandas, include opportunistic pathogens. The giant panda is an endangered species, classified as vulnerable by the World Wildlife Foundation. Continuous monitoring for the emergence of antimicrobial resistance (AMR) among bacterial isolates from giant pandas is vital not only for their protection but also for public health. RESULTS: A total of 166 E. coli, 68 Enterobacter spp., 116 K. pneumoniae and 117 Enterococcus spp. isolates were collected from fecal samples of 166 giant pandas. In the antimicrobial susceptibility tests, 144 E. coli isolates, 66 Enterobacter spp. isolates, 110 K. pneumoniae isolates and 43 Enterococcus spp. isolates were resistant to at least one antimicrobial. The resistant isolates carried antimicrobial resistance genes (ARGs), including sul3, blaTEM, blaSHV and tetA. The differences in the prevalence of the bla types implied that the genetic basis for ß-lactam resistance among the E. coli, Enterobacter spp. and K. pneumoniae isolates was different. The strain K. pneumoniae K85 that was resistant to sixteen antimicrobials was selected for whole genome sequencing. The genome contained Col440I, IncFIBK and IncFIIK plasmids and altogether 258 ARGs were predicted in the genome; 179 of the predicted ARGs were efflux pump genes. The genetic environment of the ß-lactamase genes blaCTX-M-3 and blaTEM-1 in the K. pneumoniae K85 genome was relatively similar to those in other sequenced K. pneumoniae genomes. In comparing the giant panda age groups, the differences in the resistance rates among E. coli, K. pneumoniae and Enterobacter spp. isolates suggested that the infections in giant pandas of different age should be treated differently. CONCLUSIONS: Antimicrobial resistance was prevalent in the bacterial isolates from the giant pandas, implying that the gut bacteria may pose serious health risks for captive giant pandas. The resistance genes in the genome of K. pneumoniae K85 were associated with insertion sequences and integron-integrase genes, implying a potential for the further spread of the antimicrobial resistance.

The Fecal and Serum Metabolomics of Giant Pandas Based on Untargeted Metabolomics.

Little is comprehensively known or understood about giant panda fecal and serum metabolites, which could serve as important indicators of the physiological metabolism of giant pandas. Therefore, we determined the contents of fecal and serum metabolites of giant pandas based on an untargeted metabolome. Four hundred and 955 metabolites were detected in the feces and serum of giant panda, respectively. Glycerophospholipid and choline metabolism were the main metabolic pathways in feces and serum. A significant correlation between the gut microbiota and fecal metabolites was found (P < 0.01). Fecal metabolites were not greatly affected by the age or gender of giant pandas, but serum metabolites were significantly affected by age and gender. The majority of different metabolites caused by age were higher in serum of younger giant pandas, including fatty acids, lipids, metabolites of bile acids, and intermediate products of vitamin D3. The majority of different metabolites caused by gender included fatty acids, phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE). A separate feeding diet should be considered according to different ages and genders of giant panda. Therefore, our results could provide helpful suggestions to further protect captive giant pandas.

Diversity and Composition of Gut Bacterial Community in Giant Panda with Anorexia.

The giant panda (GP) is the most precious animal in China. Gastrointestinal tract disease, especially associated with dysbiosis of gut microbiota, is the leading cause of death in GPs. Here, we performed 16S rRNA high-throughput sequencing to investigate the gut microbiota of GPs having symptoms of anorexia. Results showed that gut microbiota of GP with anorexia had lower richness (Chao1 index) than the healthy GP. However, no significant differences in alpha diversity were observed. There is a significance in the microbial structure between anorexia and healthy GPs. The abundance of phylum Firmicutes (99.23% ± 7.1%), unidentified genus Clostridiales (24.75% ± 2.5%), was significantly higher in the subadult anorexia group (P < 0.01), and that of the unidentified genus Clostridiales (4.53% ± 1.2%) was also significantly higher in the adult anorexia group (P < 0.01). Weissella and Streptococcus were found to be decreased in both anorexia groups. The decreased abundance of Weissella (0.02% ± 0.0%, 0.08% ± 0.0%) and Streptococcus (73.89% ± 4.3%, 91.15% ± 7.6%) and increase in Clostridium may cause symptoms of anorexia in giant pandas. The correlation analysis indicated that there is a symbiotic relationship among Streptococcus, Leuconostoc, Weissella, and Bacillus which are classified as probiotics (r > 0.6, P < 0.05). Importantly, a negative correlation has been found between Streptococcus and unidentified_Clostridium in two groups (r > 0.6, P < 0.05). Our results suggested that Streptococcus might be used as probiotics to control the growth of Clostridium causing the anorexia.

Metagenomic analysis revealed a wide distribution of antibiotic resistance genes and biosynthesis of antibiotics in the gut of giant pandas.

BACKGROUND: The gut microbiome is essential for the host's health and serves as an essential reservoir of antibiotic resistance genes (ARGs). We investigated the effects of different factors, including the dietary shifts and age, on the functional characteristics of the giant panda's gut microbiome (GPs) through shotgun metagenome sequencing. We explored the association between gut bacterial genera and ARGs within the gut based on network analysis. RESULTS: Fecal samples (n=60) from captive juvenile, adult, and geriatric GPs were processed, and variations were identified in the gut microbiome according to different ages, the abundance of novel ARGs and the biosynthesis of antibiotics. Among 667 ARGs identified, nine from the top ten ARGs had a higher abundance in juveniles. For 102 ARGs against bacteria, a co-occurrence pattern revealed a positive association for predominant ARGs with Streptococcus. A comparative KEGG pathways analysis revealed an abundant biosynthesis of antibiotics among three different groups of GPs, where it was more significantly observed in the juvenile group. A co-occurrence pattern further revealed a positive association for the top ten ARGs, biosynthesis of antibiotics, and metabolic pathways. CONCLUSION: Gut of GPs serve as a reservoir for novel ARGs and biosynthesis of antibiotics. Dietary changes and age may influence the gut microbiome's functional characteristics; however, it needs further studies to ascertain the study outcomes.

Diet, habitat environment and lifestyle conversion affect the gut microbiomes of giant pandas.

Gut microbiota (GM) are important for the health of giant pandas (GPs), in addition to the utilization of bamboo in their diets. However, it is not fully understood how diet, habitat environment and lifestyle contribute to the composition of GM in GP. Consequently, we evaluated how dietary changes, habitat environment conversions and lifestyle shifts influence the GM of GPs using high-throughput sequencing and genome-resolved metagenomics. The GM of GPs were more similar when their hosts exhibited the same diet. High fiber diets significantly increased the diversity and decreased the richness of gut bacterial communities alone or interacted with the age factor (p < 0.05). The abundances of Streptococcus, Pseudomonas, Enterococcus, Lactococcus, Acinetobacter, and Clostridium significantly increased during diet conversion process (Non-parametric factorial Kruskal-Wallis sum-rank test, LDA > 4). Reconstruction of 60 metagenome-assembled-genomes (MAGs) indicated that these bacteria were likely responsible for bamboo digestion via gene complements involved in cellulose, hemicellulose, and lignin degradation. While habitat environment may play a more important role in shaping the GM of GP, lifestyle can also greatly affect bacterial communities. The GM structure in reintroduced GPs notably converged to that of wild pandas. Importantly, the main bacterial genera of wild GPs could aid in lignin degradation, while those of reintroduced GPs were related to cellulose and hemicellulose digestion. Streptococcus, Pseudomonas, Enterococcus, Lactococcus, Acinetobacter, and Clostridium may contribute to lignocellulose digestion in GP. The results revealed that diet conversion, habitat environment and lifestyle could remarkably influence the GM of GP. In addition, results suggested that increasing the ability of lignin degradation with GM may aid to change the GM of reintroduced pandas to resemble those of wild pandas.

First Steps toward the Giant Panda Metabolome Database: Untargeted Metabolomics of Feces, Urine, Serum, and Saliva by 1H NMR.

Differences in the concentration of metabolites in the biofluids of animals closely reflect their physiological diversities. In order to set the basis for a metabolomic atlas for giant panda (Ailuropoda melanoleuca), we characterized the metabolome of healthy giant panda feces (23), urine (16), serum (6), and saliva (4) samples by means of 1H NMR. A total of 107 metabolites and a core metabolome of 12 metabolites was quantified across the four biological matrices. Through univariate analysis followed by robust principal component analysis, we were able to describe how the molecular profile observed in giant panda urine and feces was affected by gender and age. Among the molecules modified by age in feces, fucose plays a peculiar role because it is related to the digestion of bamboo's hemicellulose, which is considered as the main source of energy for giant panda. A metagenomic investigation directed toward this molecule showed that its concentration was indeed positively related to the two-component system pathway and negatively related to the amino sugar and nucleotide sugar metabolism pathway. Such work is meant to provide a robust framework for further -omics research studies on giant panda to accelerate our understanding of the interaction of giant panda with its natural environment.

Comparative Analysis of Gut Microbiota Among the Male, Female and Pregnant Giant Pandas (Ailuropoda Melanoleuca).

The giant panda (GP) was the most endangered species in China, and gut microbiota plays a vital role in host health. To determine the differences of the gut microbiota among the male, female and pregnant GPs, a comparative analysis of gut microbiota in GPs was carried out by 16S rRNA and ITS high-throughput sequencing. In 16S rRNA sequencing, 435 OTUs, 17 phyla and 182 genera were totally detected. Firmicutes (53.6%) was the predominant phylum followed by Proteobacteria (37.8%) and Fusobacteria (7.1%). Escherichia/Shigella (35.9%) was the most prevalent genus followed by Streptococcus (25.9%) and Clostridium (11.1%). In ITS sequencing, 920 OTUs, 6 phyla and 322 genera were also detected. Ascomycota (71.3%) was the predominant phylum followed by Basidiomycota (28.4%) and Zygomycota (0.15%). Purpureocillium (4.4%) was the most prevalent genus followed by Cladosporium (2.5%) and Pezicula (2.4%). Comparative analysis indicated that the male GPs harbor a higher abundance of phylum Firmicutes than female GPs with the contribution from genus Streptococcus. Meanwhile, the female GPs harbor a higher abundance of phylum Proteobacteria than male GPs with the contribution from genus Escherichia/ Shigella. In addition, the shift in bacteria from female to pregnant GPs indicated that phylum Firmicutes increased significantly with the contribution from Clostridium in the gut, which may provide an opportunity to study possible associations with low reproduction of the GPs.

Metagenomic Analysis of Bacteria, Fungi, Bacteriophages, and Helminths in the Gut of Giant Pandas.

To obtain full details of gut microbiota, including bacteria, fungi, bacteriophages, and helminths, in giant pandas (GPs), we created a comprehensive microbial genome database and used metagenomic sequences to align against the database. We delineated a detailed and different gut microbiota structures of GPs. A total of 680 species of bacteria, 198 fungi, 185 bacteriophages, and 45 helminths were found. Compared with 16S rRNA sequencing, the dominant bacterium phyla not only included Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria but also Cyanobacteria and other eight phyla. Aside from Ascomycota, Basidiomycota, and Glomeromycota, Mucoromycota, and Microsporidia were the dominant fungi phyla. The bacteriophages were predominantly dsDNA Myoviridae, Siphoviridae, Podoviridae, ssDNA Inoviridae, and Microviridae. For helminths, phylum Nematoda was the dominant. In addition to previously described parasites, another 44 species of helminths were found in GPs. Also, differences in abundance of microbiota were found between the captive, semiwild, and wild GPs. A total of 1,739 genes encoding cellulase, ß-glucosidase, and cellulose ß-1,4-cellobiosidase were responsible for the metabolism of cellulose, and 128,707 putative glycoside hydrolase genes were found in bacteria/fungi. Taken together, the results indicated not only bacteria but also fungi, bacteriophages, and helminths were diverse in gut of giant pandas, which provided basis for the further identification of role of gut microbiota. Besides, metagenomics revealed that the bacteria/fungi in gut of GPs harbor the ability of cellulose and hemicellulose degradation.

The normal vaginal and uterine bacterial microbiome in giant pandas (Ailuropoda melanoleuca).

While the health effects of the colonization of the reproductive tracts of mammals by bacterial communities are widely known, there is a dearth of knowledge specifically in relation to giant panda microbiomes. In order to investigate the vaginal and uterine bacterial diversity of healthy giant pandas, we used high-throughput sequence analysis of portions of the 16S rRNA gene, based on samples taken from the vaginas (GPV group) and uteri (GPU group) of these animals. Results showed that the four most abundant phyla, which contained in excess of 98% of the total sequences, were Proteobacteria (59.2% for GPV and 51.4% for GPU), Firmicutes (34.4% for GPV and 23.3% for GPU), Actinobacteria (5.2% for GPV and 14.0% for GPU) and Bacteroidetes (0.3% for GPV and 10.3% for GPU). At the genus level, Escherichia was most abundant (11.0%) in the GPV, followed by Leuconostoc (8.7%), Pseudomonas (8.0%), Acinetobacter (7.3%), Streptococcus (6.3%) and Lactococcus (6.0%). In relation to the uterine samples, Janthinobacterium had the highest prevalence rate (20.2%), followed by Corynebacterium (13.2%), Streptococcus (19.6%), Psychrobacter (9.3%), Escherichia (7.5%) and Bacteroides (6.2%). Moreover, both Chao1 and abundance-based coverage estimator (ACE) species richness indices, which were operating at the same sequencing depth for each sample, demonstrated that GPV had more species richness than GPU, while Simpson and Shannon indices of diversity indicated that GPV had the higher bacterial diversity. These findings contribute to our understanding of the potential influence abnormal reproductive tract microbial communities have on negative pregnancy outcomes in giant pandas.

NORMAL VAGINAL BACTERIAL FLORA OF GIANT PANDAS (AILUROPODA MELANOLEUCA) AND THE ANTIMICROBIAL SUSCEPTIBILITY PATTERNS OF THE ISOLATES.

To study the typical vaginal bacterial flora of giant pandas (Ailuropoda melanoleuca), we took vaginal swabs for the sake of bacterial isolation, from 24 healthy female giant pandas. A total of 203 isolates were identified, representing a total of 17 bacterial species. The most common bacteria isolated were Lactobacillus spp. (54.2%, 13/24), followed by Staphylococcus epidermidis (41.7%, 10/24) and Escherichia coli (33.3%, 8/24). Some opportunistic pathogenic bacteria, such as Peptostreptococcus spp., Klebsiella pneumoniae , and Proteus mirabilis , were also isolated but showed no pathology. Antimicrobial susceptibility testing of aerobic bacterial isolates was performed with the disk diffusion method. Of the 152 isolates, resistance was most frequently observed with chloramphenicol (17.8%), followed by tetracycline (14.5%), ciprofloxacin (12.5%), streptomycin (11.8%), and florfenicol (11.8%), whereas 7.2% were multidrug resistant. This is the first report of the normal culturable vaginal bacterial flora of giant pandas and the antimicrobial susceptibility patterns of the isolates.

NORMAL VAGINAL BACTERIAL FLORA OF GIANT PANDAS (AILUROPODA MELANOLEUCA) AND THE ANTIMICROBIAL SUSCEPTIBILITY PATTERNS OF THE ISOLATES.

In order to study the typical vaginal bacterial flora of giant pandas (Ailuropoda melanoleuca), we took vaginal swabs for the sake of bacterial isolation, from 24 healthy female giant pandas. A total of 203 isolates were identified, representing a total of 17 bacterial species. The most common bacteria isolated were Lactobacillus spp. (54.2%, 13 of 24), followed by Staphylococcus epidermidis (41.7%, 10 of 24) and Escherichia coli (33.3%, 8 of 24). Some opportunistic pathogenic bacteria, such as Peptostreptococcus spp., Klebsiella pneumoniae, and Proteus mirabilis, were also isolated but showed no pathology. Antimicrobial susceptibility testing of aerobic bacterial isolates was performed with disk diffusion method. Of the 152 isolates, resistance was most frequently observed with chloramphenicol (17.8%), followed by tetracycline (14.5%), ciprofloxacin (12.5%), streptomycin (11.8%), and florfenicol (11.8%), while 7.2% were multidrug resistant. This is the first report of the normal vaginal culturable bacterial flora of giant pandas, followed by the antimicrobial susceptibility patterns of the isolates.