Core microbes identification and synthetic microbiota construction for the production of Xiaoqu light-aroma Baijiu.

Baijiu production has relied on natural inoculated Qu as a starter culture, causing the unstable microbiota of fermentation grains, which resulted in inconsistent product quality across batches. Therefore, revealing the core microbes and constructing a synthetic microbiota during the fermentation process was extremely important for stabilizing product quality. In this study, the succession of the microbial community was analyzed by high-throughput sequencing technology, and ten core microbes of Xiaoqu light-aroma Baijiu were obtained by mathematical statistics, including Acetobacter, Bacillus, Lactobacillus, Weissella, Pichia,Rhizopus, Wickerhamomyces, Issatchenkia, Saccharomyces, and Kazachstania. Model verification showed that the core microbiota significantly affected the composition of non-core microbiota (P < 0.01) and key flavor-producing enzymes (R > 0.8, P < 0.01), thus significantly affecting the flavor of base Baijiu. Simulated fermentation validated that the core microbiota can reproduce the fermentation process and quality of Xiaoqu light-aroma Baijiu. The succession of bacteria was mainly regulated by acidity and ethanol, while the fungi, especially non-Saccharomyces cerevisiae, were mainly regulated by the initial dominant bacteria (Acetobacter, Bacillus, and Weissella). This study will play an important role in the transformation of Xiaoqu light-aroma Baijiu fermentation from natural fermentation to controlled fermentation and the identification of core microbes in other fermented foods.

Developing defined starter culture for reproducible profile of flavour compound in Chinese xiaoqu baijiu fermentation.

Defined starter cultures, containing selected microbes could reduce the complexity of natural starter, are beneficial for controllable food fermentations. However, there are challenges in identifying key microbiota and constructing synthetic microbiota for traditional food fermentations. Here, we aimed to develop a defined starter culture for reproducible profile of flavour compounds, using Chinese Xiaoqu Baijiu fermentation as a case. We classified all microbes into 4 modules using weighted correlation network analysis. Module 3 presented significant correlations with flavour compounds (P < 0.05) and the highest gene abundance related with flavour compound production. 13 dominant species in module 3 were selected for mixed culture fermentation, and each species was individually deleted to analyse the effect on flavour compound production. Ten species, presenting significant effects (P < 0.05) on flavour compound production, were selected for developing the starter culture, including Rhizopus oryzae, Rhizopus microsporus, Saccharomyces cerevisiae, Pichia kudriavzevii, Wickerhamomyces anomalus, Lactobacillus acetotolerans, Levilactobacillus brevis, Weissella paramesenteroides, Pediococcus acidilactici, and Leuconostoc pseudomesenteroides. After optimising the structure of the starter culture, the profile similarity of flavour compounds produced by the starter culture reached 81.88% with that by the natural starter. This work indicated feasibility of reproducible profile of flavour compounds with defined starter culture for food fermentations.

Compound heterozygous B3GALNT2 mutations in a fetus with encephalocele: A case report.

An encephalocele is a congenital malformation characterized by protrusion of the intracranial contents through a cranial defect. We report that a fetus of a pregnant mother who had two consecutive pregnancies with ultrasound-detected encephalocele carried compound heterozygous variants in B3GALNT2 NM_152490.5:c.[1423C > T (p.Gln475Ter)]; [261-2A > G] of maternal and paternal origins, respectively, as confirmed by exome sequencing followed by Sanger sequencing validation. The present case implies that mutations in B3GALNT2, a well-known dystroglycanopathy causative gene, may result in a phenotype of neural tube defect, providing new insights into the clinical spectrum of B3GALNT2-related disorders. Our study may contribute to prenatal screening/diagnosis and genetic counseling of congenital brain malformations.

Epidemiological investigation of Entamoeba in wild rhesus macaques in China: A novel ribosomal lineage and genetic differentiation of Entamoeba nuttalli.

Wild rhesus macaques are a potential source of zoonotic parasites for humans, and Entamoeba spp. are common intestinal parasites. To investigate the prevalence of Entamoeba in wild rhesus macaques in China and explore the genetic differentiation of the potentially pathogenic species Entamoeba nuttalli, a total of 276 fecal samples from five populations at high altitudes (HAG, 2,800-4,100 m above sea level) and four populations at low altitudes (LAG, 5-1,000 m above sea level) were collected. PCR methods based on the ssrRNA gene were used to detect Entamoeba infection. Genotyping of E. nuttalli was performed based on six tRNA-linked short tandem repeat (STR) loci for further genetic analyses. The results revealed that Entamoeba infection (69.2%) was common in wild rhesus macaques in China, especially in LAG which had a significantly higher prevalence rate than that in HAG (P < 0.001). Three zoonotic species were identified: Entamoeba chattoni (60.9%) was the most prevalent species and distributed in all the populations, followed by Entamoeba coli (33.3%) and Entamoeba nuttalli (17.4%). In addition, a novel Entamoeba ribosomal lineage named RL13 (22.8%) was identified, and phylogenetic analysis revealed a close genetic relationship between RL13 and Entamoeba. hartmanni. Genotyping of E. nuttalli obtained 24 genotypes from five populations and further analysis showed E. nuttalli had a high degree of genetic differentiation (FST > 0.25, Nm < 1) between the host populations. The result of analysis of molecular variance (AMOVA) revealed that observed genetic differences mainly originate from differences among populations (FST = 0.91). Meanwhile, the phylogenetic tree showed that these genotypes of E. nuttalli were clustered according to geographical populations, indicating a significant phylogeographic distribution pattern. Considering the potential pathogenicity of E. nuttalli, attention should be paid to its risk of zoonotic transmission.

Antimicrobial resistance spectrum and virulence characterization of Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis isolated from asymptomatic and diarrheal rhesus monkeys.

This study aims to deepen our understanding of the drug resistance and virulence characterization among gut bacteria in asymptomatic and diarrheal captive rhesus macaques (RMs). A total of 31 samples, including 8 asymptomatic RMs, 10 diarrheal RMs, and 1 dead RM, were collected from a breeding base in Sichuan, China, for bacterial isolation. As a result, Escherichia coli (n = 23), Klebsiella (n = 22), Proteus mirabilis (n = 10), Enterococcus (n = 10), Salmonella (n = 2), and Staphylococcus (n = 2) were isolated. All isolates were subjected to antimicrobial susceptibility testing and whole-genome sequencing, among which some E. coli, K. pneumoniae, and P. mirabilis were subjected to the Galleria mellonella and mice infection testing. The antimicrobial resistance rates of levofloxacin, enrofloxacin, and cefotaxime in diarrhea-associated isolates were higher than those of asymptomatic isolates. Consistent with the antimicrobial resistance phenotype, diarrheal isolates had a higher prevalence rate to qnrS1, blaTEM-1B and blaCTX-M-27 than asymptomatic isolates. Furthermore, compared with asymptomatic isolates, diarrheal isolates demonstrated a higher pathogenic potential against larvae and mice. Additionally, sequence types (STs) 14179-14181 in E. coli and ST 625 and ST 630-631 in Klebsiella aerogenes were firstly characterized. Our evidence underscores the considerable challenge posed by high rates of bacterial drug resistance in the effective treatment of diarrheal RMs.

Aroma Compound Changes in the Jiangxiangxing Baijiu Solid-State Distillation Process: Description, Kinetic Characters and Cut Point Selection.

Solid-state distillation is a distinctive process for extracting the baijiu aroma compounds that determine the flavor character of baijiu. In this study, the changes in various chemical properties of the aroma compounds in three classical Jiangxiangxing baijiu fermented grain distillation processes were analyzed. The changes in the aroma compounds in the instantaneous distillates were quantified and correlation analyzes were conducted. The results showed that the effect of the aroma compounds was greater than the differences between the fermented grains. Eleven representative aroma compounds were selected to develop the kinetic models describing two opposing changes. For the regulation of the Jiangxiangxing baijiu aroma compounds, their recovery rates were calculated using a kinetic model. A comprehensive comparison of the recovery rates of the characteristic aroma and other aroma compounds at different cut-off values revealed that the optimum recovery rate of the characteristic aroma of Jiangxiangxing baijiu 2,3,5,6-tetramethylpyrazine was 14.53% at cut-off values of 3.9 and 19.8 min. In this study, representative changes in the aroma compounds and the selection of cut-off values to regulate the baijiu distillation aroma were proposed.

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.

Insights into antibiotic and heavy metal resistance interactions in Escherichia coli isolated from livestock manure and fertilized soil.

Heavy metal and antibiotic-resistant bacteria from livestock feces are ecological and public health problems. However, the distribution and relationships of antibiotic resistance genes (ARGs), heavy metal resistance genes (HMRGs), and virulence factors (VFs) and their transmission mechanisms remain unclear. Therefore, we investigated the resistance of Escherichia coli, the prevalence of its ARGs, HMRGs, and VFs, and their transmission mechanisms in livestock fresh feces (FF), composted feces (CF), and fertilized soil (FS). In total, 99.54% (n = 221) and 91.44% (n = 203) of E. coli were resistant to at least one antibiotic and one heavy metal, respectively. Additionally, 72.52% (n = 161) were multi-drug resistant (MDR), of which Cu-resistant E. coli accounted for 72.67% (117/161). More than 99.34% (88/89) of E. coli carried multidrug ARGs, VFs, and the Cu resistance genes cueO and cusABCRFS. The Cu resistance genes cueO and cusABCRFS were mainly located on chromosomes, and cueO and cusF were positively associated with HMRGs, ARGs, and VFs. The Cu resistance genes pcoABCDRS were located on the plasmid pLKYL-P02 flanked by ARGs in PF18C from FF group and on chromosomes flanked by HMRGs in SAXZ1-1 from FS group. These results improved our understanding of bacterial multidrug and heavy metal resistance in the environment.

AIM2 participates in house dust mite (HDM)-induced epithelial dysfunctions and ovalbumin (OVA)-induced allergic asthma in infant mice.

Objective: Allergen sensitization and high rates of concomitant allergic diseases are characteristic of severe pediatric asthma. The present study was aimed to explore the mechanism of allergic asthma via bioinformatics and experiment investigation.Methods: The GSE27011 dataset contained the expression profiles of normal and pediatric asthma white blood cells was downloaded for analyzing the different expression genes and function enrichment. The allergic asthma model in infant mice was established by ovalbumin (OVA) stimulation. The cellular model was established by house dust mite (HDM)-stimulation in human bronchial epithelial cells. The absent in melanoma 2 (AIM2) knockdown was achieved by intranasal lentivirus injection or cell infection. The bronchoalveolar lavage fluid (BALF) was collected for cell counting and ELISA assessment of cytokines. Lung tissues were collected for HE staining and immunohistochemical (IHC) staining. Real-time PCR and immunoblotting were used for the determination of key gene expressions in mouse and cell models.Results: upregulation of AIM2 gene expression was observed in pediatric asthma patients based on GSE27011 and OVA-induced infant mouse allergic asthma model. AIM2 knockdown ameliorated OVA caused elevation in airway hyper-responsiveness (AHR), elevation in cell quantities (eosinophils, neutrophils, lymphocytes), and levels of cytokines (IL-4, IL-13, TNF-α, and OVA-specific IgE) in BALF. Moreover, AIM2 knockdown relieved OVA-caused histopathological alterations in mouse lungs, up-regulation of AIM2 levels, and NOD1 and receptor-interacting protein 2 (RIP2) protein levels, as well as p65 phosphorylation. In the cell model, AIM2 knockdown partially ameliorated HDM-induced epithelial dysfunctions by promoting cell viability, down-regulating inflammatory cytokines levels, and decreasing the protein levels of AIM2, NOD1, RIP2, and phosphorylated p65.Conclusion: AIM2 participates in HDM-induced epithelial dysfunctions and OVA-induced allergic asthma progression. AIM2 could be a promising target for pediatric allergic asthma treatment regimens, which warrants further in vivo investigations.

Exploring the impact of initial moisture content on microbial community and flavor generation in Xiaoqu baijiu fermentation.

Moisture is essential in microbiota succession and flavor formation during baijiu fermentation. However, it remains unknown how moisture content affects microbiota, metabolism, and their relationship. Here, we compared the difference in volatiles, microbiota characteristics, and potential functions with different initial moisture contents (50 %, 55 %, 60 %, 65 %, 70 %). Results showed that the ratio of ethyl acetate to ethyl lactate and total volatile compounds content increased as the moisture content was elevated from 50 % to 70 %. As increasing moisture content, fermentation system microbiota dominated by Lactobacillus was formed more rapidly. Lactobacillus, Dekkera, and Pediococcus were positively correlated with moisture, promoting the production of propanol, acetic acid, butyric acid, and 2-butanol. The complexity and stability of ecological networks enhanced as moisture content increased (R2 = 0.94, P = 0.004). Our study revealed that moisture-drive microbiota was a critical contributor to flavor formation, providing the theoretical basis for moisture control to regulate flavor compounds.

Gastrointestinal microbiome, resistance genes, and risk assessment of heavy metals in wild giant pandas.

The gastrointestinal microbiome (GM) of giant panda (GP) plays an important role in food utilization and health and is also an essential reservoir of resistance genes. Currently, little knowledge is available on the GM, acid resistance genes (AcRGs), antibiotic resistance genes (ARGs), metal resistance genes (MRGs), and mobile genetic elements (MGEs) in wild GPs. We sampled the gastrointestinal tract of a dead GP and explored the composition and function of GM and resistance genes through cryo-scanning electron microscopy, metagenomic sequencing, and genome-resolved metagenomics. The concentration of metals in the gastrointestinal lumen, feces, bamboo, and soil was measured by inductively coupled plasma mass spectrometry. Results showed that the composition of the microbiota varied in different gastrointestinal regions. Fecal microbiota was highly associated with small intestinal and colonic microbes. The lignocellulosic cross-linked structure of bamboo was destroyed in the stomach initially and destroying degree increased from stomach to anus. Reconstruction of metagenome-assembled-genomes confirmed that core GM, e.g., Streptococcus, Clostridium, Lactococcus, Leuconostoc, and Enterococcus, carried genes encoding the lignocellulose degradation enzyme. There were no significant differences of resistance genes between gastrointestinal and fecal samples, except MGEs. Multidrug and multi-metal resistance genes were predominant in all samples, while the transposase gene tnpA was the major type of MGE. Significant correlations were observed among the abundance of GM, resistance genes, and MGEs. Gastrointestinal and fecal mercury and chromium were the main metals influencing GM and resistance genes. The content of gastrointestinal and fecal metals was significantly associated with the presence of the same metals in bamboo, which could pose a threat to the health of wild GPs. This study characterized the gastrointestinal microbiome of wild GPs, providing new evidence for the role of the gastrointestinal microbiome in degrading lignocellulose from bamboo and highlighting the urgent need to monitor metal levels in soil and bamboo.

Class 1 integron carrying qacEΔ1 gene confers resistance to disinfectant and antibiotics in Salmonella.

Salmonella has presented increasingly alarming rates of antimicrobial resistance believed to be a result of a high prevalence of integrons. It is speculated that disinfectant-resistant isolates are due to the expression of qacEΔ1, an efflux pump located in the 3' conserved sequence (3'CS) of class 1 integrons. With this concern, we tested the antibiotic and disinfectant resistance of 581 Salmonella strains collected from different sources, and characterized their integron structures. Gene expression and induction experiments were also performed. Results showed that Salmonella have high resistance to antimicrobials, especially to sulfonamides (SAs, 78.83 %), tetracyclines (TCs, 75.04 %) and benzalkonium chloride (BC, 87.26 %). The multi-drug resistance (MDR) frequency reached up to 63.17 %, and the prevalence of intI1 was 45.78 %. Molecular characterization of class 1 integrons exhibited nine different gene cassette arrays, of these, dfrA12-orf-aadA2 (n = 75), EstX (n = 25) and aadA2 (n = 14) were the most frequent. Importantly, 74.06 % of intI1-positive isolates were carrying qacEΔ1-sul1 genes in the 3'CS. This study also demonstrated that phenotypic resistance to both antibiotics and disinfectants was significantly correlated with the emergence of intI1 (p < 0.05). 91.37 % of qacEΔ1-sul1 positive Salmonella were found with disinfectant resistance. Additionally, expression of qacEΔ1 gene in Escherichia coli confirmed qacEΔ1 is predominantly involved in conferring disinfectant resistance. Disinfectant induction experiments further implicated qacEΔ1 in disinfectant resistance. RT-qPCR revealed a disinfectant-mediated increase in the relative expression of antibiotic-resistant genes (ARGs), aadA2 and dfrA12 on the integron, and efflux pump genes (mdtH and acrD) indicating that disinfectant could trigger co or cross-resistance. Therefore, our study confirmed that using disinfectant could provide selection pressure for strains with acquired resistance to antibiotics, providing new insights into the public health impact of Salmonella and guide continued efforts in antimicrobial stewardship and prevention of antibiotic resistance.

Effects of environmental disinfection on microbial population and resistance genes: A case study of the microecology within a panda enclosure.

Widespread use of disinfectants raises concerns over their involvement in altering microbial communities and promoting antimicrobial resistance. This study explores the influence of disinfection protocols on microbial populations and resistance genes within an isolated enclosure environment and in the gut of giant pandas (GPs) held within. Samples of panda feces, air conditioning ducts, soil and bamboo were collected before and after disinfection. High-throughput sequencing characterized the microbial flora of GP gut and environmental microbes inside the artificial habitat. Microbial cultures showed that Escherichia coli (34.6%), Enterococcus (15.4%) and other pathogenic bacteria deposited in feces and the enclosure. Isolates exhibit a consistent resistance to disinfectant, with the greatest resistance shown to cyanuric acid, and the lowest to glutaraldehyde-dodecyl dimethyl ammonium bromide (GD-DDAB) and dodecyl dimethyl ammonium bromide (DDAB). The total number of the culturable bacteria in soil and bamboo were significantly diminished after disinfection but increased in the gut. After disinfection, the richness (Chao1 index) of environment samples increased significantly (P < 0.05), while the richness in gut decreased significantly (P < 0.05). Ten genera showed significant change in feces after disinfection. Metagenome sequencing showed that 126 types of virulence genes were present in feces before disinfection and 37 in soil. After disinfection, 110 virulence genes localized in feces and 53 in soil. Eleven virulence genes including ECP and T2SS increased in feces. A total of 182 antibiotic resistance genes (ARGs) subtypes, potentially conferring resistance to 20 classes of drugs, were detected in the soils and feces, with most belonging to efflux pump protein pathways. After disinfection, the number of resistance genes increased both in gut and soil, which suggests disinfection protocols increase the number of resistance pathways. Our study shows that the use of disinfectants helps to shape the microbial community of GPs and their habitat, and increases populations of resistant strain bacteria.

Gut microbiota enhance energy accumulation of black-necked crane to cope with impending migration.

Less is known about the role of gut microbiota in overwintering environmental adaptation in migratory birds. Here, we performed metagenomic sequencing on fresh fecal samples (n = 24) collected during 4 periods of overwintering (Dec: early; Jan: middle I; Feb: middle II; Mar: late) to characterize gut microbial taxonomic and functional characteristics of black-necked crane (Grus nigricollis). The results demonstrated no significant change in microbial diversity among overwintering periods. Analysis of compositions of microbiomes with bias correction (ANCOM-BC) determined 15 Proteobacteria species enriched in late overwintering period. Based on previous reports, these species are associated with degradation of chitin, cellulose, and lipids. Meanwhile, fatty acid degradation and betalain biosynthesis pathways are enriched in late overwintering period. Furthermore, metagenomic binning obtained 91 high-quality bins (completeness >70% and contamination <10%), 5 of which enriched in late overwintering period. Carnobacterium maltaromaticum, unknown Enterobacteriaceae, and Yersinia frederiksenii have genes for chitin and cellulose degradation, acetate, and glutamate production. Unknown Enterobacteriaceae and Y. frederiksenii hold genes for synthesis of 10 essential amino acids required by birds, and the latter has genes for γ-aminobutyrate production. C. maltaromaticum has genes for pyridoxal synthesis. These results implied the gut microbiota is adapted to the host diet and may help black-necked cranes in pre-migratory energy accumulation by degrading the complex polysaccharide in their diet, supplying essential amino acids and vitamin pyridoxal, and producing acetate, glutamate, and γ-aminobutyrate that could stimulate host feeding. Additionally, enriched Proteobacteria also encoded more carbohydrate-active enzymes (CAZymes) and antibiotic resistance genes (ARGs) in late overwintering period. KEY POINTS: • Differences in gut microbiota function during overwintering period of black-necked cranes depend mainly on changes in core microbiota abundance • Gut microbiota of black-necked crane adapted to the diet during overwintering period • Gut microbiota could help black-necked cranes to accumulate more energy in the late overwintering period.

Diet and high altitude strongly drive convergent adaptation of gut microbiota in wild macaques, humans, and dogs to high altitude environments.

Animal gut microbiota plays an indispensable role in host adaptation to different altitude environments. At present, little is known about the mechanism of animal gut microbiota in host adaptation to high altitude environments. Here, we selected wild macaques, humans, and dogs with different levels of kinship and intimate relationships in high altitude and low altitude environments, and analyzed the response of their gut microbiota to the host diet and altitude environments. Alpha diversity analysis found that at high altitude, the gut microbiota diversity of wild macaques with more complex diet in the wild environments is much higher than that of humans and dogs with simpler diet (p < 0.05), and beta diversity analysis found that the UniFrac distance between humans and dogs was significantly lower than between humans and macaques (p < 0.05), indicating that diet strongly drive the convergence of gut microbiota among species. Meanwhile, alpha diversity analysis found that among three subjects, the gut microbiota diversity of high altitude population is higher than that of low altitude population (ACE index in three species, Shannon index in dog and macaque and Simpson index in dog, p < 0.05), and beta diversity analysis found that the UniFrac distances among the three subjects in the high altitude environments were significantly lower than in the low altitude environments (p < 0.05). Additionally, core shared ASVs analysis found that among three subjects, the number of core microbiota in high altitude environments is higher than in low altitude environments, up to 5.34 times (1,105/207), and the proportion and relative abundance of the core bacteria types in each species were significantly higher in high altitude environments than in low altitude environments (p < 0.05). The results showed that high altitude environments played an important role in driving the convergence of gut microbiota among species. Furthermore, the neutral community model trial found that the gut microbiota of the three subjects was dispersed much more at high altitude than at low altitude, implying that the gut microbiota convergence of animals at high altitudes may be partly due to the microbial transmission between hosts mediated by human activities.

Extensive prevalence and significant genetic differentiation of Blastocystis in high- and low-altitude populations of wild rhesus macaques in China.

BACKGROUND: Blastocystis is a common intestinal protist with a wide range of hosts. Thus far, 38 subtypes have been identified. In recent years, wild animals have been confronted with habitat fragmentation as well as an increasing risk of zoonotic disease transmission due to human disturbance. Only limited data are available on Blastocystis infection and subtype distribution in wild rhesus macaques in China. The aim of the present study was to investigate the prevalence and genetic diversity of Blastocystis in nine wild rhesus macaque populations in China. METHODS: A total of 276 faecal samples were collected from five high-altitude populations (high-altitude group [HAG]; 2800-4100 m a.s.l.) and four low-altitude populations (low-altitude group [LAG]; 5-1000 m a.s.l) of rhesus macaques. PCR-based analysis, using a new primer pair for the amplification of a 1690-bp sequence of the small subunit ribosomal RNA (SSU rRNA) gene, was used for prevalence and genetic diversity analysis. RESULT: Analysis of faecal samples revealed that Blastocystis infection was common in rhesus macaques, with an infection positivity rate of 80.1% (n = 221/276 samples). There was no significant difference (P = 0.121) in positivity rate between the LAG (84.3%) and HAG (76.8%). Overall, 33 haplotypes were obtained and classified into four subtypes (STs), of which three were potentially zoonotic subtypes (ST1, 29.7%; ST2, 16.7%; ST3, 31.9%) and one that was first identified in this study and named ST39 (12.0%). The STs were distributed differently among the rhesus macaque populations, except for ST3, which was found in all populations. Phylogenetic analyses revealed two major divergent clades of ST3 for the HAG and LAG. Genetic diversity analysis showed a high genetic diversity of ST3 (haplotype diversity: 0.846; nucleotide diversity: 0.014) in the rhesus macaques, but a high genetic differentiation (FST > 0.25) and a low gene flow (Nm = 0.09) between the HAG and LAG. CONCLUSION: Our study, which is the first investigation on Blastocystis infection in multiple wild rhesus macaque populations in China, indicates a potential risk of zoonotic transmission of Blastocystis in the study areas. Blastocystis ST3 showed high genetic diversity in wild rhesus macaques and significant genetic differentiation between the HAG and LAG. Our results provide fundamental information on the genetic diversity and prevalence of Blastocystis in wild rhesus macaque populations.

Assembly of novel microbial genomes from gut metagenomes of rhesus macaque (Macaca mulatta).

Rhesus macaque (RM, Macaca mulatta), as an important model animal, commonly suffers from chronic diarrheal disease, challenging the breeding of RMs. Gut microbiomes play key roles in maintaining intestinal health of RMs. However, it is still unclear about more features of gut microbiome as responsible for intestinal health of RMs. In this study, we performed de novo assembly of metagenome-assembled genomes (MAGs) based on fecal metagenomes from chronic diarrheal RMs and asymptomatic individuals. In total of 731 non-redundant MAGs with at least 80% completeness were reconstructed in this study. More than 97% MAGs were novel genomes compared with more than 250,000 reference genomes. MAGs of Campylobacter and Helicobacteraceae from RM guts mainly carried flagella-associated virulence genes and chemotaxis-associated virulence genes, which might mediate motility and adhesion of bacteria. Comparing to MAGs of Campylobacter from humans, distributions and functions of these MAGs of Campylobacter from RMs exhibited significant differences. Most members of Bacteroidota, Spirochaetota, Helicobacteraceae, Lactobacillaceae and Anaerovibrio significantly decreased in guts of chronic diarrhea RMs. More than 92% MAGs in this study were not contained in 2,985 MAGs previously reported from other 22 non-human primates (NHPs), expanding the microbial diversity in guts of NHPs. The distributions and functions of gut microbiome were prominently influenced by host phylogeny of NHPs. Our results could help to more clearly understand about the diversity and function of RMs gut microbiome.

Screening of ß -damascenone-producing strains in light-flavor Baijiu and its production optimization via response surface methodology.

As a C13-norisoprenoid aroma substance, ß-damascenone is a highly important aromatic compound and an active constituent. The purpose of this study was to investigate the change law of ß-damascenone during the light-flavor Baijiu brewing process, and screen the indigenous microbial strains that produce this compound and optimize fermentation parameters for improving ß-damascenone production using a statistical approach. In this project, Wickerhamomyces anomalus YWB-1 exhibited the highest producing activity of ß-damascenone. Fermentation conditions were optimized for ß-damascenone production using a one-factor-at-a-time (OFAT) approach. A Plackett-Burman design was subsequently adopted to assess the effects of initial pH, incubation temperature, inoculum size, fermentation period, and original Brix degree. Analysis of variance (ANOVA) showed that the correlation coefficient (R 2) of the executive model was 0.9795, and this value was significant (p < 0.05). Three significant variables were optimized at three different coded levels using a Box-Behnken design (BBD) of response surface methodology (RSM). Here, 7.25 µg/L ß-damascenone was obtained under the following optimum conditions: initial pH of 3.31, original Brix degree of 10.53%, and fermentation period of 52.13 h. The yield was increased 3.02-fold compared with that obtained under unoptimized conditions. This information is conducive to the control of flavor production by regulating variable parameters in Baijiu fermentation.

The gut microbiome and antibiotic resistome of chronic diarrhea rhesus macaques (Macaca mulatta) and its similarity to the human gut microbiome.

BACKGROUND: Chronic diarrhea is a common disease causing morbidity and mortality of captive rhesus macaques (RMs, Macaca mulatta). Chronic diarrhea in RMs is typically characterized by long-term diarrhea and a weak response to antibiotic treatment. Diarrhea is also a common disease in humans and can cause death. However, the etiology of about half of diarrheal cases of humans is still unclear. Therefore, we performed shotgun metagenomic sequencing to characterize the differences in the gut microbiome and resistome of chronic diarrhea RMs and asymptomatic individuals. RESULTS: Our results showed Lactobacillus spp. (mainly L. johnsonii, L. reuteri and L. amylovorus) were significantly depleted in chronic diarrhea RM guts compared to asymptomatic individuals (5.2 vs 42.4%). Functional annotation of genes suggested these Lactobacillus spp. carried genes involved in the adhesion of intestinal epithelial cells and production of bacteriocin. Chronic diarrhea RM guts also had a significantly greater abundance of many other gut bacteria, including mucin-degrading bacteria and opportunistic pathogens. The metabolic pathways of chronic diarrhea RM gut microbiome were enriched in aerobactin biosynthesis, while the metabolic pathways of asymptomatic RM gut microbiome were enriched in the production of short-chain fatty acids (SCFAs). Chronic diarrhea RM guts had a significantly greater abundance of antibiotic resistance genes (ARGs), such as ermF, aph(3')-IIIa, ermB, and floR. The strains isolated from feces and tissue fluid of chronic diarrhea RMs had higher resistance rates to the majority of tested antibiotics, but not cephamycin and carbapenem antibiotics. Gut microbial composition comparisons showed that several captive nonhuman primate (NHP) guts were more similar to the guts of humans with a non-westernized diet than humans with a westernized diet. Chronic diarrhea RM gut microbiome was strikingly similar to rural-living humans with diarrhea and humans with a non-westernized diet than asymptomatic RMs. CONCLUSIONS: Our results suggested chronic diarrhea significantly altered the composition and metabolic pathways of the RM gut microbiome. The frequent use of antibiotics caused antibiotic resistance in chronic diarrhea RM gut microbiome with serious consequences for individual treatment and survival. The findings of this study will help us to improve the effective prevention and treatment of diarrhea in RMs. Video Abstract.

Comparative metagenomics analysis reveals how the diet shapes the gut microbiota in several small mammals.

The gut microbiomes of the host are large and complex communities, which helps to maintain homeostasis, improves digestive efficiency, and promotes the development of the immune system. The small mammals distributed in Sichuan Province are the most popular species for biodiversity research in Southwest China. However, the effects of different diets on the structure and function of the gut microbial community of these small mammals are poorly understood. In this study, whole-metagenome shotgun sequencing has been used to analyze the composition and functional structures of the gut microbiota of seven small mammals in Laojunshan National Nature Reserve, Sichuan Province, China. Taxonomic classification revealed that the most abundant phyla in the gut of seven small mammals were Bacteroides, Proteobacteria, and Firmicutes. Moreover, Hafnia, Lactobacillus, and Yersinia were the most abundant genus in the gut microbiomes of these seven species. At the functional level, we annotated a series of KEGG functional pathways, six Cazy categories, and 46,163 AROs in the gut microbiomes of the seven species. Comparative analysis found that the difference in the gut microbiomes between the Soricidea and Muridae concentrated on the increase in the F/B (Firmicutes/Bacteroides) ratio in the Soricidea group, probably driven by the high-fat and -calorie digestive requirements due to their insectivorous diet. The comparative functional profiling revealed that functions related to metabolism and carbohydrates were significantly more abundant in Muridae group, which may be attributed to their high carbohydrate digestion requirements caused by their herbivorous diet. These data suggested that different diets in the host may play an important role in shaping the gut microbiota, and lay the foundation for teasing apart the influences of heritable and environmental factors on the evolution of gut microbial communities.