Complex Microbial Communities Research Articles

Metatranscriptomics-guided genome-scale metabolic modeling of microbial communities

Multi-omics data integration via mechanistic models of metabolism is a scalable and flexible framework for exploring biological hypotheses in microbial systems. However, although most microorganisms are unculturable, such multi-omics modeling is limited to isolate microbes or simple synthetic communities. Here, we developed an approach for modeling microbial activity and interactions that leverages the reconstruction of metagenome-assembled genomes and associated genome-centric metatranscriptomes. At its core, we designed a method for condition-specific metabolic modeling of microbial communities through the integration of metatranscriptomic data. Using this approach, we explored the behavior of anaerobic digestion consortia driven by hydrogen availability and human gut microbiota dysbiosis associated with Crohn's disease, identifying condition-dependent amino acid requirements in archaeal species and a reduced short-chain fatty acid exchange network associated with disease, respectively. Our approach can be applied to complex microbial communities, allowing a mechanistic contextualization of multi-omics data on a metagenome scale.

Recovery and Analysis of Long-Read Metagenome-Assembled Genomes.

The development of long-read nucleic acid sequencing is beginning to make very substantive impact on the conduct of metagenome analysis, particularly in relation to the problem of recovering the genomes of member species of complex microbial communities. Here we outline bioinformatics workflows for the recovery and characterization of complete genomes from long-read metagenome data and some complementary procedures for comparison of cognate draft genomes and gene quality obtained from short-read sequencing and long-read sequencing.

Identification of Age and Gender Specific Bacteria in Human Saliva through Next- Generation Sequencing

Characterization of human saliva through Next-Generation Sequencing (NGS) has emerged as a valuable tool forunderstanding the complex microbial communities residing in the oral cavity. This study aims to investigate theage and gender-based variations in the salivary microbiome using NGS technology. Saliva samples were collectedfrom a diverse population representing females among below 45 Vs above 45 and males among below 45 Vsabove 45. The DNA from the samples was extracted, and theV3-V4 region of the 16S rRNA gene was amplifiedfor NGS analysis. The obtained sequences were processed and analysed using bioinformatics tools to determinethe microbial composition and diversity. Preliminary results revealed distinct microbial profiles, indicating thepotential influence of age and gender on the salivary microbiome.Further research is warranted to explore additional factors and expand the scope of habit-based analysis in salivabasedmicrobial characterization through NGS.

A View of Anaerobic Digestion: Microbiology, Advantages and Optimization

Anaerobic digestion is an efficient technology widely used in biodegradable waste treatment, in which both pollution control and energy recovery could be achieved. Sewage sludge generated by waste water treatment plants is an ideal candidate for anaerobic digestion, for the total amount of sludge could be considerably reduced during the digestion process with a large sum of biogas produced as energy resource for heating, electricity or vehicle power. Although complex microbial community structures and interrelationships in anaerobic digestion process lead to the difficult of managing and maintaining the system, there has been various advanced methods developed to optimize the anaerobic digestion technology. Co-digestion of feedstocks from different sources and pre-treatments with chemical agents, physical methods or biological organics are all proved to be effective methods for enhancement of anaerobic digestion technology in the aspects of pathogen destruction and biogas production.

한국 전통발효식품과 마이크로바이옴

Traditional food fermentation involves complex microbial community and their interactions. Despite the beneficial properties of these microbes, their complexity remains a challenge in predicting the key players involved in fermentation. Technological advancements in meta-omics approaches such as metataxonomics, metagenomics, and metatranscriptomics enable scientists to identify the total microbial community and their functions during different stages of fermentation. In the present study, we have discussed different Korean fermented foods and key microbes during fermentation, identified using next generation sequencing (NGS) techniques.

The Use of Natural Methods to Control Foodborne Biofilms.

Biofilms are large aggregates of various species of bacteria or other microorganisms tightly attached to surfaces through an intricate extracellular matrix. These complex microbial communities present quite the challenge in the food processing industry, as conditions such as raw meats and diverse food product content in contact with workers, drains, machinery, and ventilation systems, make for prime circumstances for contamination. Adding to the challenge is the highly resistant nature of these biofilm growths and the need to keep in mind that any antimicrobials utilized in these situations risk health implications with human consumption of the products that are being processed in these locations. For that reason, the ideal means of sanitizing areas of foodborne biofilms would be natural means. Herein, we review a series of innovative natural methods of targeting foodborne biofilms, including bacteriocins, bacteriophages, fungi, phytochemicals, plant extracts, essential oils, gaseous and aqueous control, photocatalysis, enzymatic treatments, and ultrasound mechanisms.

Difficulties Of Enterobacteriaceae Genome Annotation In Deciphering Gastrointestinal Microbiome Datasets Obtained By 16S RRNA Gene Amplicon Sequencing

Sequencing of the 16S rRNA gene amplicon is the cornerstone of the method for studying diverse bacteria in complex microbial communities. However, its use is complicated by an error rate of 10–17% when annotating 16S rRNA gene sequences. In our study, we examined the degree of accuracy of the taxonomic database of Enterobacteriaceae, compiled using the SILVA 132 reference database and a previously obtained dataset, viz. the microbiome of the gastrointestinal tract in adolescents with normal body weight and obesity. Material and Methods — In this study, previously obtained 16S rRNA gene amplicon sequencing data were used, and the deciphering was carried out using the QIIME2 2019.4 platform. Phylogenetic analysis was performed using MEGA X software. Results — Phylogenetic analysis of this family based on the studied V3–V4 fragment was hampered by polyphyly among some genera, and for half of the variants of the amplicon sequences it was not possible to clarify their genus. Statistical analysis did not reveal significant differences between the samples. Conclusion — Although the average values of bacterial genera in the studied groups intuitively differed from each other, statistical analysis did not reveal significant differences between the samples. However, it can be assumed that a more detailed study of taxonomic diversity, taking into account factors, such as enterotype, duration of breastfeeding and family history, may reveal differences in the frequency distribution.

Confocal Laser Scanning Microscopy Approach to Investigate Plant-Fungal Interactions.

Plants interact with a broad range of microorganisms, such as bacteria and fungi. In plant roots, complex microbial communities participate in plant nutrition and development as well as in the protection against stresses. The establishment of the root microbiota is a dynamic process in space and time regulated by abiotic (e.g., edaphic, climate, etc.) and biotic factors (e.g., host genotype, root exudates, etc.). In the last 20years, the development of metabarcoding surveys, based on high-throughput next-generation sequencing methods, identified the main drivers of microbial community structuration. However, identification of plant-associated microbes by sequencing should be complemented by imaging techniques to provide information on the micrometric spatial organization and its impact on plant-fungal and fungal-fungal interactions. Laser scanning confocal microscopy can provide both types of information and is now used to investigate communities of endophytic, endomycorrhizal, and ectomycorrhizal fungi. In this chapter, we present a protocol enabling the detection of fungal individuals and communities associated to the plant root system.

Interplay between rhizospheric Pseudomonas chlororaphis strains lays the basis for beneficial bacterial consortia.

Pseudomonas chlororaphis (Pc) representatives are found as part of the rhizosphere-associated microbiome, and different rhizospheric Pc strains frequently perform beneficial activities for the plant. In this study we described the interactions between the rhizospheric Pc strains PCL1601, PCL1606 and PCL1607 with a focus on their effects on root performance. Differences among the three rhizospheric Pc strains selected were first observed in phylogenetic studies and confirmed by genome analysis, which showed variation in the presence of genes related to antifungal compounds or siderophore production, among others. Observation of the interactions among these strains under lab conditions revealed that PCL1606 has a better adaptation to environments rich in nutrients, and forms biofilms. Interaction experiments on plant roots confirmed the role of the different phenotypes in their lifestyle. The PCL1606 strain was the best adapted to the habitat of avocado roots, and PCL1607 was the least, and disappeared from the plant root scenario after a few days of interaction. These results confirm that 2 out 3 rhizospheric Pc strains were fully compatible (PCL1601 and PCL1606), efficiently colonizing avocado roots and showing biocontrol activity against the fungal pathogen Rosellinia necatrix. The third strain (PCL1607) has colonizing abilities when it is alone on the root but displayed difficulties under the competition scenario, and did not cause deleterious effects on the other Pc competitors when they were present. These results suggest that strains PCL1601 and PCL1606 are very well adapted to the avocado root environment and could constitute a basis for constructing a more complex beneficial microbial synthetic community associated with avocado plant roots.

GgMOB: Elucidation of genomic conjugative features and associated cargo genes across bacterial genera using genus-genus mobilization networks.

Horizontal gene transfer mediated by conjugation is considered an important evolutionary mechanism of bacteria. It allows organisms to quickly evolve new phenotypic properties including antimicrobial resistance (AMR) and virulence. The frequency of conjugation-mediated cargo gene exchange has not yet been comprehensively studied within and between bacterial taxa. We developed a frequency-based network of genus-genus conjugation features and candidate cargo genes from whole-genome sequence data of over 180,000 bacterial genomes, representing 1,345 genera. Using our method, which we refer to as ggMOB, we revealed that over half of the bacterial genomes contained one or more known conjugation features that matched exactly to at least one other genome. Moreover, the proportion of genomes containing these conjugation features varied substantially by genus and conjugation feature. These results and the genus-level network structure can be viewed interactively in the ggMOB interface, which allows for user-defined filtering of conjugation features and candidate cargo genes. Using the network data, we observed that the ratio of AMR gene representation in conjugative versus non-conjugative genomes exceeded 5:1, confirming that conjugation is a critical force for AMR spread across genera. Finally, we demonstrated that clustering genomes by conjugation profile sometimes correlated well with classical phylogenetic structuring; but that in some cases the clustering was highly discordant, suggesting that the importance of the accessory genome in driving bacterial evolution may be highly variable across both time and taxonomy. These results can advance scientific understanding of bacterial evolution, and can be used as a starting point for probing genus-genus gene exchange within complex microbial communities that include unculturable bacteria. ggMOB is publicly available under the GNU licence at https://ruiz-hci-lab.github.io/ggMOB/.

Novel Virus Identification through Metagenomics: A Systematic Review.

Metagenomic Next Generation Sequencing (mNGS) allows the evaluation of complex microbial communities, avoiding isolation and cultivation of each microbial species, and does not require prior knowledge of the microbial sequences present in the sample. Applications of mNGS include virome characterization, new virus discovery and full-length viral genome reconstruction, either from virus preparations enriched in culture or directly from clinical and environmental specimens. Here, we systematically reviewed studies that describe novel virus identification through mNGS from samples of different origin (plant, animal and environment). Without imposing time limits to the search, 379 publications were identified that met the search parameters. Sample types, geographical origin, enrichment and nucleic acid extraction methods, sequencing platforms, bioinformatic analytical steps and identified viral families were described. The review highlights mNGS as a feasible method for novel virus discovery from samples of different origins, describes which kind of heterogeneous experimental and analytical protocols are currently used and provides useful information such as the different commercial kits used for the purification of nucleic acids and bioinformatics analytical pipelines.

Microbial metabolic transformation and antioxidant activity evaluation of polyphenols in kombucha

Kombucha is a kind of traditional fermented sugared tea rich in polyphenols. Although the health effects of tea polyphenols (TPPs) have been extensively studied, the biotransformation of the TPPs during kombucha fermentation was not fully elucidated. In this study, the polyphenol transformation capacity of microbial strains and the synthetic microbial communities during the tea broth fermentation were investigated. Firstly, six microbial species demonstrated different polyphenol transformation abilities: Acetobacter pasteurianus, Zygosaccharomyces bailii and Debaryomyce hansenii could increase the contents of epigallocatechin gallate (EGCG), the most abundant components of TPPs. Z. bailii and Acetobacter xylinum could enhance the concentration of TPPs with low molecular weight. Then, four synthetic microbial communities (SMC) consisting of different species were constructed to ferment the black tea broth. When different strains coexisted, their interaction could further enhance kombucha's phenols transformation and antioxidant activity. Specifically, the SMC, constructed by Z. bailii, L. plantarum and D. hansenii, could enhance the EGCG content significantly in tea broth and the total phenols and flavonoid contents. Meanwhile, the antioxidant activities of kombucha fermented by this SMC were found to be relatively high among different fermented tea broth groups. Besides, it is demonstrated that A. pasteurianus was a strong competitor in the tea broth environment. This bacterium could decrease the number of viable yeast cells by producing more acid. This study demonstrated that the construction of suitable synthetic microbial communities would help reveal the phenolic metabolic characteristics of complex microbial communities and obtain customizable phenolic combinations for kombucha products.

Exploiting Conserved Quorum Sensing Signals in Streptococcus mutans and Streptococcus pneumoniae

Bacterial species of the Streptococcus genera are considered either commensal bacteria or potential pathogens, according to their metabolic evolution and production of quorum sensing (QS)-controlled virulence factors. S. mutans, in particular, has become one of the best-studied examples of bacteria that are able to get along or cheat commensal species, even of the same genera. S. mutans and S. pneumoniae share homolog QS pathways and a competence stimulating peptide (CSP) for regulating bacteriocin production. Intriguingly, the abundance of S. pneumoniae and S. mutans alternates in complex microbial communities, thus opening the role for the fratricide communication of homolog QS systems. Since the inhibition of the QS has been proposed in treating bacterial infections, in this study, we designed and synthesized analogs of S. pneumoniae CSP with precise residual modifications. We reported that S. pneumoniae CSP analogs reduced the expression of genes involved in the QS of S. mutans and biofilm formation without affecting bacterial growth. The CSP analogs inhibited bacteriocin production in S. mutans, as reported by co-cultures with commensal bacteria of the oral cavity. The peptide CSP1AA, bearing substitutions in the residues involved in QS receptor recognition and activation, reported the most significant quorum-quenching activities. Our findings provide new insights into specific chemical drivers in the CSP sequences controlling the interconnection between S. mutans and S. pneumoniae. We think that the results reported in this study open the way for new therapeutic interventions in controlling the virulence factors in complex microbial communities such as the oral microbiota.

Latent functional diversity may accelerate microbial community responses to temperature fluctuations.

How complex microbial communities respond to climatic fluctuations remains an open question. Due to their relatively short generation times and high functional diversity, microbial populations harbor great potential to respond as a community through a combination of strain-level phenotypic plasticity, adaptation, and species sorting. However, the relative importance of these mechanisms remains unclear. We conducted a laboratory experiment to investigate the degree to which bacterial communities can respond to changes in environmental temperature through a combination of phenotypic plasticity and species sorting alone. We grew replicate soil communities from a single location at six temperatures between 4°C and 50°C. We found that phylogenetically and functionally distinct communities emerge at each of these temperatures, with K-strategist taxa favored under cooler conditions and r-strategist taxa under warmer conditions. We show that this dynamic emergence of distinct communities across a wide range of temperatures (in essence, community-level adaptation) is driven by the resuscitation of latent functional diversity: the parent community harbors multiple strains pre-adapted to different temperatures that are able to 'switch on' at their preferred temperature without immigration or adaptation. Our findings suggest that microbial community function in nature is likely to respond rapidly to climatic temperature fluctuations through shifts in species composition by resuscitation of latent functional diversity.

Real-time monitoring of biofilm formation using a noninvasive impedance-based method

Biofilms are complex three-dimensional microbial communities that adhere to a variety of surfaces and interact with their surroundings. Because of the dynamic nature of biofilm formation, establishing a uniform technique for quantifying and monitoring biofilm volume, shape, and features in real-time is challenging. Herein, we describe a noninvasive electrochemical impedance approach for real-time monitoring of dental plaque-derived multispecies biofilm growth on a range of substrates. A working equation relating electrochemical impedance to live biofilm volume has been developed that is applicable to all three surfaces examined, including glass, dental filling resin, and Ca2+-releasing resin composites. Impedance changes of 2.5 %, 35 %, 50 %, and 65 % correlated to biofilm volumes of 0.10 ± 0.01, 16.9 ± 2.2, 29.7 ± 2.3, and 38.6 ± 2.8 µm3/μm2, respectively. We discovered that glass, dental filling resin, and Ca2+-releasing dental composites required approximately 3.5, 4.5, and 6 days, respectively, to achieve a 50 % change in impedance. The local pH change at the biofilm-substrate interfaces also monitored with potentiometry pH microsensor, and pH change varied according to biofilm volume. This impedance-based technique can be a useful analytical method for monitoring the growth of biofilms on a variety of substrates in real-time. Therefore, this technique may be beneficial for examining antibacterial properties of novel biomaterials.

Reproducible changes in the anorexia nervosa gut microbiota following inpatient therapy remain distinct from non-eating disorder controls

ABSTRACT The composition of the gut microbiota in patients with anorexia nervosa (AN), and the ability of this microbial community to influence the host, remains uncertain. To achieve a broader understanding of the role of the intestinal microbiota in patients with AN, we collected fecal samples before and following clinical treatment at two geographically distinct eating disorder units (Center of Excellence for Eating Disorders [UNC-CH] and ACUTE Center for Eating Disorders [Denver Health]). Gut microbiotas were characterized in patients with AN, before and after inpatient treatment, and in non-eating disorder (non-ED) controls using shotgun metagenomic sequencing. The impact of inpatient treatment on the AN gut microbiota was remarkably consistent between eating disorder units. Although weight in patients with AN showed improvements, AN microbiotas post-treatment remained distinct from non-ED controls. Additionally, AN gut microbiotas prior to treatment exhibited more fermentation pathways and a lower ability to degrade carbohydrates than non-ED controls. As the intestinal microbiota can influence nutrient metabolism, our data highlight the complex microbial communities in patients with AN as an element needing further attention post inpatient treatment. Additionally, this study defines the effects of renourishment on the AN gut microbiota and serves as a platform to develop precision nutrition approaches to potentially mitigate impediments to recovery.

Characterization of the core microflora and nutrient composition in packaged pasteurized milk products during storage

Pasteurized milk contains complex microbial communities affected by sterilization and storage conditions. This complex microflora may be the possible reason that pasteurized dairy products are highly prone to spoilage. In this study, packaged pasteurized milk products collected from dairy processing factories in China were stored at 0, 4, 10, 15, and 25 ºC for 0−15 days and subjected to microbial identification using high-throughput sequencing. Accordingly, 6 phyla and 44 genera were identified as the dominant microbiota. Moreover, the changes in nutritional composition of the pasteurized milk, including in 16 free amino acids, 7 taste values, and 8 chemical constituents, were analyzed using principal component and multi-factor analyses. The Pearson correlation analysis identified Pseudomonas, Aeromonas, Paenibacillus, and Serratia genera as the core functional microbiota that significantly affects the nutritional composition of pasteurized milk. Hence, the results provide a comprehensive understanding of the safety and shelf-life of stored pasteurized milk.

Optimization of soil microbial fuel cell for sustainable bio-electricity production: combined effects of electrode material, electrode spacing, and substrate feeding frequency on power generation and microbial community diversity

BackgroundMicrobial fuel cells (MFCs) are among the leading research topics in the field of alternative energy sources due to their multifunctional potential. However, their low bio-energy production rate and unstable performance limit their application in the real world. Therefore, optimization is needed to deploy MFCs beyond laboratory-scale experiments. In this study, we investigated the combined influence of electrode material (EM), electrode spacing (ES), and substrate feeding interval (SFI) on microbial community diversity and the electrochemical behavior of a soil MFC (S-MFC) for sustainable bio-electricity generation.ResultsTwo EMs (carbon felt (CF) and stainless steel/epoxy/carbon black composite (SEC)) were tested in an S-MFC under three levels of ES (2, 4, and 8 cm) and SFI (4, 6, and 8 days). After 30 days of operation, all MFCs achieved open-circuit voltage in the range of 782 + 12.2 mV regardless of the treatment. However, the maximum power of the SEC–MFC was 3.6 times higher than that of the CF–MFC under the same experimental conditions. The best solution, based on the interactive influence of the two discrete variables, was obtained with SEC at an ES of 4.31 cm and an SFI of 7.4 days during an operating period of 66 days. Analysis of the experimental treatment effects of the variables revealed the order SFI < ES < EM, indicating that EM is the most influential factor affecting the performance of S-MFC. The performance of S-MFC at a given ES value was found to be dependent on the levels of SFI with the SEC electrode, but this interactive influence was found to be insignificant with the CF electrode. The microbial bioinformatic analysis of the samples from the S-MFCs revealed that both electrodes (SEC and CF) supported the robust metabolism of electroactive microbes with similar morphological and compositional characteristics, independent of ES and SFI. The complex microbial community showed significant compositional changes at the anode and cathode over time.ConclusionThis study has demonstrated that the performance of S-MFC depends mainly on the electrode materials and not on the diversity of the constituent microbial communities. The performance of S-MFCs can be improved using electrode materials with pseudocapacitive properties and a larger surface area, instead of using unmodified CF electrodes commonly used in S-MFC systems.

Benchmarking second and third-generation sequencing platforms for microbial metagenomics

Shotgun metagenomic sequencing is a common approach for studying the taxonomic diversity and metabolic potential of complex microbial communities. Current methods primarily use second generation short read sequencing, yet advances in third generation long read technologies provide opportunities to overcome some of the limitations of short read sequencing. Here, we compared seven platforms, encompassing second generation sequencers (Illumina HiSeq 300, MGI DNBSEQ-G400 and DNBSEQ-T7, ThermoFisher Ion GeneStudio S5 and Ion Proton P1) and third generation sequencers (Oxford Nanopore Technologies MinION R9 and Pacific Biosciences Sequel II). We constructed three uneven synthetic microbial communities composed of up to 87 genomic microbial strains DNAs per mock, spanning 29 bacterial and archaeal phyla, and representing the most complex and diverse synthetic communities used for sequencing technology comparisons. Our results demonstrate that third generation sequencing have advantages over second generation platforms in analyzing complex microbial communities, but require careful sequencing library preparation for optimal quantitative metagenomic analysis. Our sequencing data also provides a valuable resource for testing and benchmarking bioinformatics software for metagenomics.

Effect of Inoculum Microbial Diversity in Ex Situ Biomethanation of Hydrogen.

The effects of the inoculum origin, temperature or operational changes on ex situ biomethanation by complex microbial communities have been investigated; however, it remains unclear how the diversity of the inoculum influences the process and its stability. We explored the effect of microbial diversity of four inocula (coded as PF, WW, S37 and Nrich) on methane production, process stability and the formation of volatile fatty acids as by-products. The highest methane amounts produced were 3.38 ± 0.37 mmol, 3.20 ± 0.07 mmol, 3.07 ± 0.27 mmol and 3.14 ± 0.06 mmol for PF, WW, S37 and Nrich, respectively. The highest acetate concentration was found in less diverse cultures (1679 mg L-1 and 1397 mg L-1 for S37 and Nrich, respectively), whereas the acetate concentrations remained below 30 mg L-1 in the more diverse cultures. The maximum concentration of propionate was observed in less diverse cultures (240 mg L-1 and 37 mg L-1 for S37 and Nrich cultures, respectively). The highly diverse cultures outperformed the medium and low diversity cultures in the long-term operation. Methanogenic communities were mainly composed of hydrogenotrophic methanogens in all cultures. Aceticlastic methanogenesis was only active in the highly diverse sludge community throughout the experiment. The more diverse the inocula, the more methane was produced and the less volatile fatty acids accumulated, which could be attributed to the high number of microbial functions working together to keep a stable and balanced process. It is concluded that the inoculum origin and its diversity are very important factors to consider when the biomethanation process is performed with complex microbial communities.