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September 22, 2019  |  

Bacterial microbiota and metabolic character of traditional sour cream and butter in Buryatia, Russia.

Traditional sour cream and butter are widely popular fermented dairy products in Russia for their flavor and nutrition, and contain rich microbial biodiversity, particularly in terms of lactic acid bacteria (LAB). However, few studies have described the microbial communities and metabolic character of traditional sour cream and butter. The objective of this study was to determine the bacterial microbiota and metabolic character of eight samples collected from herdsmen in Buryatia, Russia. Using single-molecule real-time (SMRT) sequencing techniques, we identified a total of 294 species and/or subspecies in 169 bacterial genera, belonging to 14 phyla. The dominant phylum was Firmicutes (81.47%) and the dominant genus was Lactococcus (59.28%). There were differences between the bacterial compositions of the sour cream and butter samples. The relative abundances of Lactococcus lactis, Lactococcus raffinolactis, and Acetobacter cibinongensis were significantly higher in sour cream than in butter, and the abundance of Streptococcusthermophilus was significantly lower in sour cream than in butter. Using a pure culture method, 48 strains were isolated and identified to represent seven genera and 15 species and/or subspecies. Among these isolates, Lactococccus lactis subsp. lactis (22.50%) was the dominant LAB species. Ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry at elevated energy was used in combination with statistical methods to detect metabolite differences between traditional sour cream and butter. A total of 27,822 metabolites were detected in all samples, and Lys-Lys, isohexanal, palmitic acid, Leu-Val, and 2′-deoxycytidine were the most dominant metabolites found in all samples. In addition, 27 significantly different metabolites were detected between the sour cream and butter samples, including short peptides, organic acids, and amino acids. Based on correlation analyses between the most prevalent bacterial species and the main metabolites in sour cream, we conclude that there may be a connection between the dominant LAB species and these metabolites. This study combined omics techniques to analyze the bacterial diversity and metabolic character of traditional sour cream and butter, and we hope that our findings will enrich species resource libraries and provide valuable resources for further research on dairy product flavor.

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September 22, 2019  |  

Lactobacillus fermentum FTDC 8312 combats hypercholesterolemia via alteration of gut microbiota.

In this study, hypercholesterolemic mice fed with Lactobacillus fermentum FTDC 8312 after a seven-week feeding trial showed a reduction in serum total cholesterol (TC) levels, accompanied by a decrease in serum low-density lipoprotein cholesterol (LDL-C) levels, an increase in serum high-density lipoprotein cholesterol (HDL-C) levels, and a decreased ratio of apoB100:apoA1 when compared to those fed with control or a type strain, L. fermentum JCM 1173. These have contributed to a decrease in atherogenic indices (TC/HDL-C) of mice on the FTDC 8312 diet. Serum triglyceride (TG) levels of mice fed with FTDC 8312 and JCM 1173 were comparable to those of the controls. A decreased ratio of cholesterol and phospholipids (C/P) was also observed for mice fed with FTDC 8312, leading to a decreased number of spur red blood cells (RBC) formation in mice. Additionally, there was an increase in fecal TC, TG, and total bile acid levels in mice on FTDC 8312 diet compared to those with JCM 1173 and controls. The administration of FTDC 8312 also altered the gut microbiota population such as an increase in the members of genera Akkermansia and Oscillospira, affecting lipid metabolism and fecal bile excretion in the mice. Overall, we demonstrated that FTDC 8312 exerted a cholesterol lowering effect that may be attributed to gut microbiota modulation. Copyright © 2017 Elsevier B.V. All rights reserved.

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September 22, 2019  |  

Design of primers for evaluation of lactic acid bacteria populations in complex biological samples.

Lactic acid bacteria (LAB) are important for human health. However, the relative abundance of LAB in complex samples, such as fecal samples, is low and their presence and diversity (at the species level) is understudied. Therefore, we designed LAB-specific primer pairs based on 16S rRNA gene consensus sequences from 443 species of LAB from seven genera. The LAB strains selected were genetically similar and known to play a role in human health. Prior to primer design, we obtained consistent sequences for the primer-binding sites by comparing the 16S rRNA gene sequences, manually identifying single-stranded primers and modifying these primers using degenerate bases. We assembled primer pairs with product sizes of >400 bp. Optimal LAB-specific primers were screened using three methods: PCR amplification, agarose gel electrophoresis and single-molecule real-time (SMRT) sequencing analysis. During the SMRT analysis procedure, we focused on sequence reads and diversity at the species level of target LAB in three fecal samples, using the universal bacterium primer 27f/1492r as a reference control. We created a phylogenetic tree to confirm the ability of the best candidate primer pair to differentiate amongst species. The results revealed that LAB-specific primer L5, with a product size of 750 bp, could generate 3222, 2552, and 3405 sequence reads from fecal Samples 1, 2, and 3. This represented 14, 13 and 10% of all target LAB sequence reads, respectively, compared with 2, 0.8, and 0.8% using the 27f/1492r primer. In addition, L5 detected LAB that were in low abundance and could not be detected using the 27f/1492r primer. The phylogenetic tree based on the alignments between the forward and reverse primer of L5 showed that species within the seven target LAB genera could be distinguished from each other, confirming L5 is a powerful tool for inferring phylogenetic relationships amongst LAB species. In conclusion, L5 is a LAB-specific primer that can be used for high-throughput sequencing and identification of taxa to the species level, especially in complex samples with relatively low LAB content. This enables further research on LAB population diversity in complex ecosystem, and on relationships between LAB and their hosts.

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September 22, 2019  |  

Accurate determination of bacterial abundances in human metagenomes using full-length 16S sequencing reads

DNA sequencing of PCR-amplified marker genes, especially but not limited to the 16S rRNA gene, is perhaps the most common approach for profiling microbial communities. Due to technological constraints of commonly available DNA sequencing, these approaches usually take the form of short reads sequenced from a narrow, targeted variable region, with a corresponding loss of taxonomic resolution relative to the full length marker gene. We use Pacific Biosciences single-molecule, real-time circular consensus sequencing to sequence amplicons spanning the entire length of the 16S rRNA gene. However, this sequencing technology suffers from high sequencing error rate that needs to be addressed in order to take full advantage of the longer sequence. Here, we present a method to model the sequencing error process using a generalized pair hidden Markov chain model and estimate bacterial abundances in microbial samples. We demonstrate, with simulated and real data, that our model and its associated estimation procedure are able to give accurate estimates at the species (or subspecies) level, and is more flexible than existing methods like SImple Non-Bayesian TAXonomy (SINTAX).

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September 22, 2019  |  

Complete genome sequence of Enterococcus durans Oregon-R-modENCODE strain BDGP3, a lactic acid bacterium found in the Drosophila melanogaster gut

Enterococcus durans Oregon-R-modENCODE strain BDGP3 was isolated from the Drosophila melanogaster gut for functional host-microbe interaction studies. The complete genome is composed of a single circular genome of 2,983,334 bp, with a G+C content of 38%, and a single plasmid of 5,594 bp. Copyright © 2017 Wan et al.

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September 22, 2019  |  

Investigating bacterial population structure and dynamics in traditional koumiss from Inner Mongolia using single molecule real-time sequencing.

Koumiss is considered as a complete dairy product high in nutrients and with medicinal properties. The bacterial communities involved in production of koumiss play a crucial role in the fermentation cycle. To reveal bacterial biodiversity in koumiss and the dynamics of succession in bacterial populations during fermentation, 22 samples were collected from 5 sampling sites and the full length of the 16S ribosomal RNA genes sequenced using single molecule real-time sequencing technology. One hundred forty-eight species were identified from 82 bacterial genera and 8 phyla. These results suggested that the structural difference in the bacterial community could be attributed to geographical location. The most significant difference in bacterial composition occurred in samples from group D compared with other groups. The sampling location of group D was distant from the city and maintained the primitive local nomadic life. The dynamics of succession in bacterial communities showed that Lactobacillus helveticus increased in abundance from 0 to 9h and reached its peak at 9h and then decreased. In contrast, Enterococcus faecalis, Enterococcus durans, and Enterococcus casseliflavus increased gradually throughout the fermentation process, and reached a maximum after 24h. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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September 22, 2019  |  

Assessment of the physicochemical properties and bacterial composition of Lactobacillus plantarum and Enterococcus faecium-fermented Astragalus membranaceus using single molecule, real-time sequencing technology.

We investigated if fermentation with probiotic cultures could improve the production of health-promoting biological compounds in Astragalus membranaceus. We tested the probiotics Enterococcus faecium, Lactobacillus plantarum and Enterococcus faecium?+?Lactobacillus plantarum and applied PacBio single molecule, real-time sequencing technology (SMRT) to evaluate the quality of Astragalus fermentation. We found that the production rates of acetic acid, methylacetic acid, aethyl acetic acid and lactic acid using E. faecium?+?L. plantarum were 1866.24?mg/kg on day 15, 203.80?mg/kg on day 30, 996.04?mg/kg on day 15, and 3081.99?mg/kg on day 20, respectively. Other production rates were: polysaccharides, 9.43%, 8.51%, and 7.59% on day 10; saponins, 19.6912?mg/g, 21.6630?mg/g and 20.2084?mg/g on day 15; and flavonoids, 1.9032?mg/g, 2.0835?mg/g, and 1.7086?mg/g on day 20 using E. faecium, L. plantarum and E. faecium?+?L. plantarum, respectively. SMRT was used to analyze microbial composition, and we found that E. faecium and L. plantarum were the most prevalent species after fermentation for 3 days. E. faecium?+?L. plantarum gave more positive effects than single strains in the Astragalus solid state fermentation process. Our data demonstrated that the SMRT sequencing platform is applicable to quality assessment of Astragalus fermentation.

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September 22, 2019  |  

Extensive horizontal gene transfer in cheese-associated bacteria.

Acquisition of genes through horizontal gene transfer (HGT) allows microbes to rapidly gain new capabilities and adapt to new or changing environments. Identifying widespread HGT regions within multispecies microbiomes can pinpoint the molecular mechanisms that play key roles in microbiome assembly. We sought to identify horizontally transferred genes within a model microbiome, the cheese rind. Comparing 31 newly sequenced and 134 previously sequenced bacterial isolates from cheese rinds, we identified over 200 putative horizontally transferred genomic regions containing 4733 protein coding genes. The largest of these regions are enriched for genes involved in siderophore acquisition, and are widely distributed in cheese rinds in both Europe and the US. These results suggest that HGT is prevalent in cheese rind microbiomes, and that identification of genes that are frequently transferred in a particular environment may provide insight into the selective forces shaping microbial communities.

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September 22, 2019  |  

Role of clinicogenomics in infectious disease diagnostics and public health microbiology.

Clinicogenomics is the exploitation of genome sequence data for diagnostic, therapeutic, and public health purposes. Central to this field is the high-throughput DNA sequencing of genomes and metagenomes. The role of clinicogenomics in infectious disease diagnostics and public health microbiology was the topic of discussion during a recent symposium (session 161) presented at the 115th general meeting of the American Society for Microbiology that was held in New Orleans, LA. What follows is a collection of the most salient and promising aspects from each presentation at the symposium. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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September 22, 2019  |  

Cow-to-mouse fecal transplantations suggest intestinal microbiome as one cause of mastitis.

Mastitis, which affects nearly all lactating mammals including human, is generally thought to be caused by local infection of the mammary glands. For treatment, antibiotics are commonly prescribed, which however are of concern in both treatment efficacy and neonate safety. Here, using bovine mastitis which is the most costly disease in the dairy industry as a model, we showed that intestinal microbiota alone can lead to mastitis.Fecal microbiota transplantation (FMT) from mastitis, but not healthy cows, to germ-free (GF) mice resulted in mastitis symptoms in mammary gland and inflammations in serum, spleen, and colon. Probiotic intake in parallel with FMT from diseased cows led to relieved mastitis symptoms in mice, by shifting the murine intestinal microbiota to a state that is functionally distinct from either healthy or diseased microbiota yet structurally similar to the latter. Despite conservation in mastitis symptoms, diseased cows and mice shared few mastitis-associated bacterial organismal or functional markers, suggesting striking divergence in mastitis-associated intestinal microbiota among lactating mammals. Moreover, an “amplification effect” of disease-health distinction in both microbiota structure and function was apparent during the cow-to-mouse FMT.Hence, dysbiosis of intestinal microbiota may be one cause of mastitis, and probiotics that restore intestinal microbiota function are an effective and safe strategy to treat mastitis.

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September 22, 2019  |  

Evaluation of bacterial contamination in raw milk, ultra-high temperature milk and infant formula using single molecule, real-time sequencing technology.

The Pacific Biosciences (Menlo Park, CA) single molecule, real-time sequencing technology (SMRT) was reported to have some advantages in analyzing the bacterial profile of environmental samples. In this study, the presence of bacterial contaminants in raw milk, UHT milk, and infant formula was determined by SMRT sequencing of the full length 16S rRNA gene. The bacterial profiles obtained at different taxonomic levels revealed clear differences in bacterial community structure across the 16 analyzed dairy samples. No indicative pathogenic bacteria were found in any of these tested samples. However, some of the detected bacterial species (e.g., Bacillus cereus, Enterococcus casseliflavus, and Enterococcus gallinarum) might potentially relate with product quality defects and bacterial antibiotic gene transfer. Although only a limited number of dairy samples were analyzed here, our data have demonstrated for the first time the feasibility of using the SMRT sequencing platform in detecting bacterial contamination. Our paper also provides interesting reference information for future development of new precautionary strategies for controlling the dairy safety in large-scale industrialized production lines. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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September 22, 2019  |  

Quantitative metaproteomics highlight the metabolic contributions of uncultured phylotypes in a thermophilic anaerobic digester.

In this study, we used multiple meta-omic approaches to characterize the microbial community and the active metabolic pathways of a stable industrial biogas reactor with food waste as the dominant feedstock, operating at thermophilic temperatures (60°C) and elevated levels of free ammonia (367 mg/liter NH3-N). The microbial community was strongly dominated (76% of all 16S rRNA amplicon sequences) by populations closely related to the proteolytic bacterium Coprothermobacter proteolyticus. Multiple Coprothermobacter-affiliated strains were detected, introducing an additional level of complexity seldom explored in biogas studies. Genome reconstructions provided metabolic insight into the microbes that performed biomass deconstruction and fermentation, including the deeply branching phyla Dictyoglomi and Planctomycetes and the candidate phylum “Atribacteria” These biomass degraders were complemented by a synergistic network of microorganisms that convert key fermentation intermediates (fatty acids) via syntrophic interactions with hydrogenotrophic methanogens to ultimately produce methane. Interpretation of the proteomics data also suggested activity of a Methanosaeta phylotype acclimatized to high ammonia levels. In particular, we report multiple novel phylotypes proposed as syntrophic acetate oxidizers, which also exert expression of enzymes needed for both the Wood-Ljungdahl pathway and ß-oxidation of fatty acids to acetyl coenzyme A. Such an arrangement differs from known syntrophic oxidizing bacteria and presents an interesting hypothesis for future studies. Collectively, these findings provide increased insight into active metabolic roles of uncultured phylotypes and presents new synergistic relationships, both of which may contribute to the stability of the biogas reactor.Biogas production through anaerobic digestion of organic waste provides an attractive source of renewable energy and a sustainable waste management strategy. A comprehensive understanding of the microbial community that drives anaerobic digesters is essential to ensure stable and efficient energy production. Here, we characterize the intricate microbial networks and metabolic pathways in a thermophilic biogas reactor. We discuss the impact of frequently encountered microbial populations as well as the metabolism of newly discovered novel phylotypes that seem to play distinct roles within key microbial stages of anaerobic digestion in this stable high-temperature system. In particular, we draft a metabolic scenario whereby multiple uncultured syntrophic acetate-oxidizing bacteria are capable of syntrophically oxidizing acetate as well as longer-chain fatty acids (via the ß-oxidation and Wood-Ljundahl pathways) to hydrogen and carbon dioxide, which methanogens subsequently convert to methane. Copyright © 2016 American Society for Microbiology.

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September 22, 2019  |  

Recent developments in using advanced sequencing technologies for the genomic studies of lignin and cellulose degrading microorganisms.

Lignin is a complex polyphenyl aromatic compound which exists in tight associations with cellulose and hemicellulose to form plant primary and secondary cell wall. Lignocellulose is an abundant renewable biomaterial present on the earth. It has gained much attention in the scientific community in recent years because of its potential applications in bio-based industries. Microbial degradation of lignocellulose polymers was well studied in wood decaying fungi. Based on the plant materials they degrade these fungi were classified as white rot, brown rot and soft rot. However, some groups of bacteria belonging to the actinomycetes, a-proteobacteria and ß-proteobacteria were also found to be efficient in degrading lignocellulosic biomass but not well understood unlike the fungi. In this review we focus on recent advancements deployed for finding and understanding the lignocellulose degradation by microorganisms. Conventional molecular methods like sequencing 16s rRNA and Inter Transcribed Spacer (ITS) regions were used for identification and classification of microbes. Recent progression in genomics mainly next generation sequencing technologies made the whole genome sequencing of microbes possible in a great ease. The whole genome sequence studies reveals high quality information about genes and canonical pathways involved in the lignin and other cell wall components degradation.

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September 22, 2019  |  

Genomics and host specialization of honey bee and bumble bee gut symbionts.

Gilliamella apicola and Snodgrassella alvi are dominant members of the honey bee (Apis spp.) and bumble bee (Bombus spp.) gut microbiota. We generated complete genomes of the type strains G. apicola wkB1(T) and S. alvi wkB2(T) (isolated from Apis), as well as draft genomes for four other strains from Bombus. G. apicola and S. alvi were found to occupy very different metabolic niches: The former is a saccharolytic fermenter, whereas the latter is an oxidizer of carboxylic acids. Together, they may form a syntrophic network for partitioning of metabolic resources. Both species possessed numerous genes [type 6 secretion systems, repeats in toxin (RTX) toxins, RHS proteins, adhesins, and type IV pili] that likely mediate cell-cell interactions and gut colonization. Variation in these genes could account for the host fidelity of strains observed in previous phylogenetic studies. Here, we also show the first experimental evidence, to our knowledge, for this specificity in vivo: Strains of S. alvi were able to colonize their native bee host but not bees of another genus. Consistent with specific, long-term host association, comparative genomic analysis revealed a deep divergence and little or no gene flow between Apis and Bombus gut symbionts. However, within a host type (Apis or Bombus), we detected signs of horizontal gene transfer between G. apicola and S. alvi, demonstrating the importance of the broader gut community in shaping the evolution of any one member. Our results show that host specificity is likely driven by multiple factors, including direct host-microbe interactions, microbe-microbe interactions, and social transmission.

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September 22, 2019  |  

Discovery of enzymes for toluene synthesis from anoxic microbial communities.

Microbial toluene biosynthesis was reported in anoxic lake sediments more than three decades ago, but the enzyme catalyzing this biochemically challenging reaction has never been identified. Here we report the toluene-producing enzyme PhdB, a glycyl radical enzyme of bacterial origin that catalyzes phenylacetate decarboxylation, and its cognate activating enzyme PhdA, a radical S-adenosylmethionine enzyme, discovered in two distinct anoxic microbial communities that produce toluene. The unconventional process of enzyme discovery from a complex microbial community (>300,000 genes), rather than from a microbial isolate, involved metagenomics- and metaproteomics-enabled biochemistry, as well as in vitro confirmation of activity with recombinant enzymes. This work expands the known catalytic range of glycyl radical enzymes (only seven reaction types had been characterized previously) and aromatic-hydrocarbon-producing enzymes, and will enable first-time biochemical synthesis of an aromatic fuel hydrocarbon from renewable resources, such as lignocellulosic biomass, rather than from petroleum.

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