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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

Metabolism of toxic sugars by strains of the bee gut symbiont Gilliamella apicola.

Social bees collect carbohydrate-rich food to support their colonies, and yet, certain carbohydrates present in their diet or produced through the breakdown of pollen are toxic to bees. The gut microbiota of social bees is dominated by a few core bacterial species, including the Gram-negative species Gilliamella apicola We isolated 42 strains of G. apicola from guts of honey bees and bumble bees and sequenced their genomes. All of the G. apicola strains share high 16S rRNA gene similarity, but they vary extensively in gene repertoires related to carbohydrate metabolism. Predicted abilities to utilize different sugars were verified experimentally. Some strains can utilize mannose, arabinose, xylose, or rhamnose (monosaccharides that can cause toxicity in bees) as their sole carbon and energy source. All of the G. apicola strains possess a manO-associated mannose family phosphotransferase system; phylogenetic analyses suggest that this was acquired from Firmicutes through horizontal gene transfer. The metabolism of mannose is specifically dependent on the presence of mannose-6-phosphate isomerase (MPI). Neither growth rates nor the utilization of glucose and fructose are affected in the presence of mannose when the gene encoding MPI is absent from the genome, suggesting that mannose is not taken up by G. apicola strains which harbor the phosphotransferase system but do not encode the MPI. Given their ability to simultaneously utilize glucose, fructose, and mannose, as well as the ability of many strains to break down other potentially toxic carbohydrates, G. apicola bacteria may have key roles in improving dietary tolerances and maintaining the health of their bee hosts.Bees are important pollinators of agricultural plants. Our study documents the ability of Gilliamella apicola, a dominant gut bacterium in honey bees and bumble bees, to utilize several sugars that are harmful to bee hosts. Using genome sequencing and growth assays, we found that the ability to metabolize certain toxic carbohydrates is directly correlated with the presence of their respective degradation pathways, indicating that metabolic potential can be accurately predicted from genomic data in these gut symbionts. Strains vary considerably in their range of utilizable carbohydrates, which likely reflects historical horizontal gene transfer and gene deletion events. Unlike their bee hosts, G. apicola bacteria are not detrimentally affected by growth on mannose-containing medium, even in strains that cannot metabolize this sugar. These results suggest that G. apicola may be an important player in modulating nutrition in the bee gut, with ultimate effects on host health. Copyright © 2016 Zheng et al.

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September 22, 2019

Automated broad range molecular detection of bacteria in clinical samples.

Molecular detection methods, such as quantitative PCR (qPCR), have found their way into clinical microbiology laboratories for the detection of an array of pathogens. Most routinely used methods, however, are directed at specific species. Thus, anything that is not explicitly searched for will be missed. This greatly limits the flexibility and universal application of these techniques. We investigated the application of a rapid universal bacterial molecular identification method, IS-pro, to routine patient samples received in a clinical microbiology laboratory. IS-pro is a eubacterial technique based on the detection and categorization of 16S-23S rRNA gene interspace regions with lengths that are specific for each microbial species. As this is an open technique, clinicians do not need to decide in advance what to look for. We compared routine culture to IS-pro using 66 samples sent in for routine bacterial diagnostic testing. The samples were obtained from patients with infections in normally sterile sites (without a resident microbiota). The results were identical in 20 (30%) samples, IS-pro detected more bacterial species than culture in 31 (47%) samples, and five of the 10 culture-negative samples were positive with IS-pro. The case histories of the five patients from whom these culture-negative/IS-pro-positive samples were obtained suggest that the IS-pro findings are highly clinically relevant. Our findings indicate that an open molecular approach, such as IS-pro, may have a high added value for clinical practice. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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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

Analyses of intestinal microbiota: culture versus sequencing.

Analyzing human as well as animal microbiota composition has gained growing interest because structural components and metabolites of microorganisms fundamentally influence all aspects of host physiology. Originally dominated by culture-dependent methods for exploring these ecosystems, the development of molecular techniques such as high throughput sequencing has dramatically increased our knowledge. Because many studies of the microbiota are based on the bacterial 16S ribosomal RNA (rRNA) gene targets, they can, at least in principle, be compared to determine the role of the microbiome composition for developmental processes, host metabolism, and physiology as well as different diseases. In our review, we will summarize differences and pitfalls in current experimental protocols, including all steps from nucleic acid extraction to bioinformatical analysis which may produce variation that outweighs subtle biological differences. Future developments, such as integration of metabolomic, transcriptomic, and metagenomic data sets and standardization of the procedures, will be discussed. © The Author 2015. Published by Oxford University Press on behalf of the Institute for Laboratory Animal Research. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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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

The human microbiome and understanding the 16S rRNA gene in translational nursing science.

As more is understood regarding the human microbiome, it is increasingly important for nurse scientists and healthcare practitioners to analyze these microbial communities and their role in health and disease. 16S rRNA sequencing is a key methodology in identifying these bacterial populations that has recently transitioned from use primarily in research to having increased utility in clinical settings.The objectives of this review are to (a) describe 16S rRNA sequencing and its role in answering research questions important to nursing science; (b) provide an overview of the oral, lung, and gut microbiomes and relevant research; and (c) identify future implications for microbiome research and 16S sequencing in translational nursing science.Sequencing using the 16S rRNA gene has revolutionized research and allowed scientists to easily and reliably characterize complex bacterial communities. This type of research has recently entered the clinical setting, one of the best examples involving the use of 16S sequencing to identify resistant pathogens, thereby improving the accuracy of bacterial identification in infection control. Clinical microbiota research and related requisite methods are of particular relevance to nurse scientists-individuals uniquely positioned to utilize these techniques in future studies in clinical settings.

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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

Next generation sequencing data of a defined microbial mock community.

Generating sequence data of a defined community composed of organisms with complete reference genomes is indispensable for the benchmarking of new genome sequence analysis methods, including assembly and binning tools. Moreover the validation of new sequencing library protocols and platforms to assess critical components such as sequencing errors and biases relies on such datasets. We here report the next generation metagenomic sequence data of a defined mock community (Mock Bacteria ARchaea Community; MBARC-26), composed of 23 bacterial and 3 archaeal strains with finished genomes. These strains span 10 phyla and 14 classes, a range of GC contents, genome sizes, repeat content and encompass a diverse abundance profile. Short read Illumina and long-read PacBio SMRT sequences of this mock community are described. These data represent a valuable resource for the scientific community, enabling extensive benchmarking and comparative evaluation of bioinformatics tools without the need to simulate data. As such, these data can aid in improving our current sequence data analysis toolkit and spur interest in the development of new tools.

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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

PCR and omics based techniques to study the diversity, ecology and biology of anaerobic fungi: Insights, challenges andopportunities.

Anaerobic fungi (phylum Neocallimastigomycota) are common inhabitants of the digestive tract of mammalian herbivores, and in the rumen, can account for up to 20% of the microbial biomass. Anaerobic fungi play a primary role in the degradation of lignocellulosic plant material. They also have a syntrophic interaction with methanogenic archaea, which increases their fiber degradation activity. To date, nine anaerobic fungal genera have been described, with further novel taxonomic groupings known to exist based on culture-independent molecular surveys. However, the true extent of their diversity may be even more extensively underestimated as anaerobic fungi continue being discovered in yet unexplored gut and non-gut environments. Additionally many studies are now known to have used primers that provide incomplete coverage of the Neocallimastigomycota. For ecological studies the internal transcribed spacer 1 region (ITS1) has been the taxonomic marker of choice, but due to various limitations the large subunit rRNA (LSU) is now being increasingly used. How the continued expansion of our knowledge regarding anaerobic fungal diversity will impact on our understanding of their biology and ecological role remains unclear; particularly as it is becoming apparent that anaerobic fungi display niche differentiation. As a consequence, there is a need to move beyond the broad generalization of anaerobic fungi as fiber-degraders, and explore the fundamental differences that underpin their ability to exist in distinct ecological niches. Application of genomics, transcriptomics, proteomics and metabolomics to their study in pure/mixed cultures and environmental samples will be invaluable in this process. To date the genomes and transcriptomes of several characterized anaerobic fungal isolates have been successfully generated. In contrast, the application of proteomics and metabolomics to anaerobic fungal analysis is still in its infancy. A central problem for all analyses, however, is the limited functional annotation of anaerobic fungal sequence data. There is therefore an urgent need to expand information held within publicly available reference databases. Once this challenge is overcome, along with improved sample collection and extraction, the application of these techniques will be key in furthering our understanding of the ecological role and impact of anaerobic fungi in the wide range of environments they inhabit.

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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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