Plants as a Source of Bacterial Resistance Modulators and Anti-Infective Agents

The spread of multidrug-resistant (MDR) strains of bacteria necessitates the discovery of new classes of antibacterials and compounds that inhibit these resistance mechanisms. At present, there are no single chemical entity plant-derived antibacterials used clinically, and this chemically diverse group deserves consideration as a source for two major reasons. First, plants have exceptional ability to produce cytotoxic agents and second there is an ecological rationale that antimicrobial natural products should be present or synthesised de novo in plants following microbial attack to protect the producer from pathogenic microbes in its environment. We have been characterising plant-derived products that are either antibacterial in their own right, or modulators of resistance in bacterial strains possessing multidrug efflux mechanisms. These efflux transporters are responsible for resistance to certain antibiotics and antiseptics and occur in strains of methicillin-resistant Staphylococcus aureus (MRSA), a major clinical problem at present. We are also investigating plant sources for compounds with activity against mycobacteria with a view to discovering drug leads with potential activity toward tuberculosis (TB) producing species. This paper will briefly review the literature on plant derived bacterial resistance modifying agents and antibacterials. Examples in this area from our own work will be given. The activities of plant-derived antibacterials show that there are many potential new classes of antibacterial agents which should undergo further cytotoxicity, microbial specificity and preclinical studies.     

Divining the Essence of Symbiosis: Insights from the Squid-Vibrio Model

Biology has a big elephant in the room. Researchers are learning that microorganisms are critical for every aspect of the biosphere''s health. Even at the scale of our own bodies, we are discovering the unexpected necessity and daunting complexity of our microbial partners. How can we gain an understanding of the form and function of these “ecosystems” that are an individual animal? This essay explores how development of experimental model systems reveals basic principles that underpin the essence of symbiosis and, more specifically, how one symbiosis, the squid-vibrio association, provides insight into the persistent microbial colonization of animal epithelial surfaces.     

Microbial scout hypothesis and microbial discovery

In this study, we examine the temporal pattern of colony appearance during cultivation experiments, and whether this pattern could inform on optimizing the process of microbial discovery. In a series of long-term cultivation experiments, we observed an expected gradual increase over time of the total number of microbial isolates, culminating in a 700-fold colony count increase at 18 months. Conventional thought suggests that long-term incubations result in a culture collection enriched with species that are slow growing or rare, may be unavailable from short-term experiments, and likely are novel. However, after we examined the phylogenetic novelty of the isolates as a function of the time of their isolation, we found no correlation between the two. The probability of discovering either a new or rare species late in the incubation matched that of species isolated earlier. These outcomes are especially notable because of their generality: observations were essentially identical for marine and soil bacteria as well as for spore formers and non-spore formers. These findings are consistent with the idea of the stochastic awakening of dormant cells, thus lending support to the scout model. The process of microbial discovery is central to the study of environmental microorganisms and the human microbiome. While long-term incubation does not appear to increase the probability of discovering novel species, the technology enabling such incubations, i.e., single-cell cultivation, may still be the method of choice. While it does not necessarily allow more species to grow from a given inoculum, it minimizes the overall isolation effort and supplies needed.     

Microbial community composition controls carbon flux across litter types in early phase of litter decomposition

Leaf litter decomposition is a major carbon input to soil, making it a target for increasing soil carbon storage through microbiome engineering. We expand upon previous findings to show with multiple leaf litter types that microbial composition can drive variation in carbon flow from litter decomposition and specific microbial community features are associated with synonymous patterns of carbon flow among litter types. Although plant litter type selects for different decomposer communities, within a litter type, microbial composition drives variation in the quantity of dissolved organic carbon (DOC) measured at the end of the decomposition period. Bacterial richness was negatively correlated with DOC quantity, supporting our hypothesis that across multiple litter types there are common microbial traits linked to carbon flow patterns. Variation in DOC abundance (i.e. high versus low DOC) driven by microbial composition is tentatively due to differences in bacterial metabolism of labile compounds, rather than catabolism of non-labile substrates such as lignin. The temporal asynchrony of metabolic processes across litter types may be a substantial impediment to discovering more microbial features common to synonymous patterns of carbon flow among litters. Overall, our findings support the concept that carbon flow may be programmed by manipulating microbial community composition.     

单细胞稳定同位素标记技术在固氮微生物中的应用研究

生物固氮是指固氮微生物将大气中氮气还原为生物可利用氨的过程,是环境中新氮的主要来源,调控初级生产力并影响氮储库的收支平衡。由于环境中大部分固氮微生物不可纯培养,不依赖培养且具有高空间分辨率水平的单细胞技术,成为研究固氮微生物的有力手段。~(15)N_2稳定同位素标记技术,以微生物对~(15)N的同化量或速率为依据,是表征微生物固氮活性的最直接手段。本文对~(15)N_2稳定同位素标记结合两种单细胞技术,即纳米二次离子质谱(Nano SIMS)和单细胞拉曼光谱,用于固氮微生物研究的最新进展进行了综述,内容包括揭示环境中高活性固氮微生物、空间分布、与其他生物的共生关系、细胞生理状态等,并进一步对近期发展的基于单细胞拉曼光谱的固氮微生物研究进行了展望。     

水圈微生物：推动地球重要元素循环的隐形巨人

正生活在水圈环境中的微生物数量巨大、遗传与代谢方式极为多样,它们驱动着地球上重要元素的循环。水圈微生物研究已经成为生命科学与地球科学的研究热点。国家自然科学基金委员会于2017年启动了"水圈微生物驱动地球元素循环的机制"重大研究计划(简称"水圈微生物"计划)。"水圈微生物"计划拟选择典型水圈生境,通过多学科交叉研究,借助新技术、新方法,揭示水圈微生物在物种、群落和生态水平驱动碳氮硫循环的机制及其环境响应,     

Unlocking the potential of metagenomics through replicated experimental design

Metagenomics holds enormous promise for discovering novel enzymes and organisms that are biomarkers or drivers of processes relevant to disease, industry and the environment. In the past two years, we have seen a paradigm shift in metagenomics to the application of cross-sectional and longitudinal studies enabled by advances in DNA sequencing and high-performance computing. These technologies now make it possible to broadly assess microbial diversity and function, allowing systematic investigation of the largely unexplored frontier of microbial life. To achieve this aim, the global scientific community must collaborate and agree upon common objectives and data standards to enable comparative research across the Earth's microbiome. Improvements in comparability of data will facilitate the study of biotechnologically relevant processes, such as bioprospecting for new glycoside hydrolases or identifying novel energy sources.     

The use of stable isotope probing techniques in bioreactor and field studies on bioremediation

Stable isotope probing (SIP) is a molecular technique that allows investigators to follow the flow of atoms in isotopically enriched molecules through complex microbial communities into metabolically active microorganisms. Thus, SIP has immense promise for discovering microorganisms responsible for ecologically important biogeochemical reactions in nature. Applications of SIP to biodegradation and bioremediation processes are still in their infancy. In the past few years, approximately a dozen biodegradation studies using SIP based on the analysis of labeled DNA, RNA or phospholipid fatty acids have been completed. Results have begun to link biomarkers (especially sequences of 16S ribosomal RNA and functional genes) to biodegradation reactions in naturally occurring microbial communities. As extensive compilations of ecologically important genotypes and phenotypes accrue, predictive abilities for contaminant metabolism in particular habitats may be achieved.     

Massively parallel sequencing of single cells by epicPCR links functional genes with phylogenetic markers

Many microbial communities are characterized by high genetic diversity. 16S ribosomal RNA sequencing can determine community members, and metagenomics can determine the functional diversity, but resolving the functional role of individual cells in high throughput remains an unsolved challenge. Here, we describe epicPCR (Emulsion, Paired Isolation and Concatenation PCR), a new technique that links functional genes and phylogenetic markers in uncultured single cells, providing a throughput of hundreds of thousands of cells with costs comparable to one genomic library preparation. We demonstrate the utility of our technique in a natural environment by profiling a sulfate-reducing community in a freshwater lake, revealing both known sulfate reducers and discovering new putative sulfate reducers. Our method is adaptable to any conserved genetic trait and translates genetic associations from diverse microbial samples into a sequencing library that answers targeted ecological questions. Potential applications include identifying functional community members, tracing horizontal gene transfer networks and mapping ecological interactions between microbial cells.     

Computational methods and challenges in analyzing intratumoral microbiome data

The human microbiome is intimately related to cancer biology and plays a vital role in the efficacy of cancer treatments, including immunotherapy. Extraordinary evidence has revealed that several microbes influence tumor development through interaction with the host immune system, that is, immuno–oncology–microbiome (IOM). This review focuses on the intratumoral microbiome in IOM and describes the available data and computational methods for discovering biological insights of microbial profiling from host bulk, single-cell, and spatial sequencing data. Critical challenges in data analysis and integration are discussed. Specifically, the microorganisms associated with cancer and cancer treatment in the context of IOM are collected and integrated from the literature. Lastly, we provide our perspectives for future directions in IOM research.     

海洋动物来源微生物的共培养研究进展

近年来,由于一些新疾病的发生和细菌耐药性的出现,微生物来源次级代谢产物的筛选重复率越来越高,微生物一些代谢基因在现有实验室条件下无法表达,所以需要发现新的微生物资源,同时找到激活微生物代谢产物基因的方法.海洋动物体内蕴含着大量的共附生微生物资源,可以产生很多具有生物活性的化合物,是潜在的药用资源.本文综述了近年来海洋动...     

High-throughput pyrosequencing used for the discovery of a novel cellulase from a thermophilic cellulose-degrading microbial consortium

Objectives

To analyze the microbial diversity and gene content of a thermophilic cellulose-degrading consortium from hot springs in Xiamen, China using 454 pyrosequencing for discovering cellulolytic enzyme resources.

Results

A thermophilic cellulose-degrading consortium, XM70 that was isolated from a hot spring, used sugarcane bagasse as sole carbon and energy source. DNA sequencing of the XM70 sample resulted in 349,978 reads with an average read length of 380 bases, accounting for 133,896,867 bases of sequence information. The characterization of sequencing reads and assembled contigs revealed that most microbes were derived from four phyla: Geobacillus (Firmicutes), Thermus, Bacillus, and Anoxybacillus. Twenty-eight homologous genes belonging to 15 glycoside hydrolase families were detected, including several cellulase genes. A novel hot spring metagenome-derived thermophilic cellulase was expressed and characterized.

Conclusions

The application value of thermostable sugarcane bagasse-degrading enzymes is shown for production of cellulosic biofuel. The practical power of using a short-read-based metagenomic approach for harvesting novel microbial genes is also demonstrated.

     

Mechanisms and recent advances in biological control mediated through the potato rhizosphere

Potato cultivation has a strategic role as a food source for the human population. Its promising future development relies on improving the control of the numerous microbial diseases that affect its growth. Numerous and recent studies on the potato rhizosphere, mycorrhizosphere and endorhiza reveal the presence of a diverse and dense microbial community. This microbial community constitutes a rich source for plant growth-promoting rhizobacteria and biocontrol agents. So far, the beneficial effects achieved are related to microbial siderophores, antibiotics, biosynthesis of surfactants and phytohormones, nutrient and spatial competition, mycoparasitism, induced systemic resistance, phage therapy, quorum quenching and construction of transgenic lines. Considering the crucial role for food and the diversity of mechanisms involved in growth promotion and microbial protection, potato constitutes a historical and accurate model in developing new biocontrol strategies.     

Dissolved Organic Matter Concentration and Quality Influences upon Structure and Function of Freshwater Microbial Communities

Past studies have suggested that the concentration and quality of dissolved organic matter (DOM) may influence microbial community structure. In this study, we cross-inoculated the bacterial communities from two streams and a dystrophic lake that varied in DOM concentration and chemistry, to yield nine fully crossed treatments. We measured dissolved organic carbon (DOC) concentration and heterotrophic microbial community productivity throughout a 72-h incubation period, characterized DOM quality by molecular weight, and determined microbial community structure at the initial and final time points. Our results indicate that all bacterial inoculate sources had similar effects upon DOC concentration and DOM quality, regardless of the DOM source. These effects included an overall decrease in DOM M _W and an initial period of DOC concentration variability between 0-24h. In contrast, microbial communities and their metabolic rates converged to profiles that reflected the DOM source upon which they were growing, regardless of the initial bacterial inoculation. The one exception was that the bacterial community from the low-concentration and low-molecular-weight DOM source exhibited a greater denaturing gradient gel electrophoresis (DGGE) band richness when grown in its own DOM source than when grown in the highest concentration and molecular weight DOM source. This treatment also exhibited a higher rate of productivity. In general, our data suggest that microbial communities are selected by the DOM sources to which they are exposed. A microbial community will utilize the low-molecular-weight (or labile) DOM sources as well as parts of the high-molecular-weight (refractory) DOM, until a community develops that can efficiently metabolize the more abundant high-molecular-weight source. This experiment examines some of the complex interactions between microbial community selection and the combined factors of DOM quality and concentration. Our data suggest that the roles of aerobic aquatic heterotrophic bacteria in carbon cycling, as well as the importance of high-molecular-weight DOM as a carbon source, may be more complex than is conventionally recognized.     

Bacterial and archaeal diversity in two hot spring microbial mats from the geothermal region of Tengchong, China

We investigated the bacterial and archaeal diversity in two hot spring microbial mats from the geothermal region of Tengchong in the Yunnan Province, China, using direct molecular analyses. The Langpu (LP) laminated mat was found by the side of a boiling pool with temperature of 60-65?°C and a pH of 8.5, while the Tengchong (TC) streamer mat consisted of white streamers in a slightly acidic (pH 6.5) hot pool outflow with a temperature of 72?°C. Four 16S rRNA gene clone libraries were constructed and restriction enzyme analysis of the inserts was used to identify unique sequences and clone frequencies. From almost 200 clones screened, 55 unique sequences were retrieved. Phylogenetic analysis showed that the LP mat consisted of a diverse bacterial population [Cyanobacteria, Chloroflexi, Chlorobia, Nitrospirae, 'Deinococcus-Thermus', Proteobacteria (alpha, beta and delta subdivisions), Firmicutes, Bacteroidetes and Actinobacteria], while the archaeal population was dominated by methanogenic Euryarchaeota and Crenarchaeota. In contrast, the TC streamer mat consisted of a bacterial population dominated by Aquificae, while the archaeal population also contained Korarchaeota as well as Crenarchaeota and methanogenic Euryarchaeota. These mats harboured clone sequences affiliated to unidentified lineages, suggesting that they are a potential source for discovering novel bacteria and archaea.     

Comparative Persistence of Subgroups of F-Specific RNA Phages in River Water

F-specific (F+) RNA phages are widely used as indicators for the presence of fecal contamination and/or enteric viruses in water, and identifying subgroups of F+ RNA phages provides an approach for microbial source tracking. Different survival characteristics of the F+ RNA phage subgroups result in a misinterpretation of their original proportion in water, thus giving misleading information when they are used for microbial source tracking. This study investigated the comparative persistence of subgroups of F+ RNA phages in river water under different conditions. Results suggested that temperature and pH are the major factors affecting the persistence of F+ RNA phages in river water, and organic substances promote phage survival. The comparative persistence patterns of subgroups of F+ RNA phages varied and may bias extrapolation of their initial proportions in surface water. Thus, the characteristics of water should be taken into consideration and the results should be carefully interpreted when F+ RNA phages are used for microbial source tracking.     

Dynamics of the Microbiota in Response to Host Infection

Longitudinal studies of the microbiota are important for discovering changes in microbial communities that affect the host. The complexity of these ecosystems requires rigorous integrated experimental and computational methods to identify temporal signatures that promote physiologic or pathophysiologic responses in vivo. Employing a murine model of infectious colitis with the pathogen Citrobacter rodentium, we generated a 2-month time-series of 16S rDNA gene profiles, and quantitatively cultured commensals, from multiple intestinal sites in infected and uninfected mice. We developed a computational framework to discover time-varying signatures for individual taxa, and to automatically group signatures to identify microbial sub-communities within the larger gut ecosystem that demonstrate common behaviors. Application of this model to the 16S rDNA dataset revealed dynamic alterations in the microbiota at multiple levels of resolution, from effects on systems-level metrics to changes across anatomic sites for individual taxa and species. These analyses revealed unique, time-dependent microbial signatures associated with host responses at different stages of colitis. Signatures included a Mucispirillum OTU associated with early disruption of the colonic surface mucus layer, prior to the onset of symptomatic colitis, and members of the Clostridiales and Lactobacillales that increased with successful resolution of inflammation, after clearance of the pathogen. Quantitative culture data validated findings for predominant species, further refining and strengthening model predictions. These findings provide new insights into the complex behaviors found within host ecosystems, and define several time-dependent microbial signatures that may be leveraged in studies of other infectious or inflammatory conditions.     

Hydrocarbon degradation capacity and population dynamics of a microbial consortium obtained using a sequencing batch reactor in the presence of molasses

A native microbial consortium capable of degrading hydrocarbons was employed as an inoculum source in a sequencing batch reactor (SBR) using molasses as a carbon source. The microbial biomass in the SBR was able to grow in the presence of molasses, degrading 88% of the reducing sugar. Moreover, the consortium produced in the SBR was capable of maintaining 75% of the capacity for biodegradation of oil with respect to the original capacity of the native microbial consortium. Monitoring of the microbial population structure was accomplished using PCR-DGGE. The results indicated that the microbial populations grown in molasses were stable during crude oil degradation, as judged by comparison to the population structure of the native microbial consortium. The results obtained demonstrated that molasses could be used as a carbon source to promote the growth of biomass with oildegrading capacity.     

Use of olive mill wastewater compost for crop production

Vast amounts of olive mill wastewaters (OMW) are produced in Mediterranean countries, where their treatment and disposal are becoming a serious environmental problem. Increasing attention has been paid to discovering a use for OMW and a wide range of technological treatments are available nowadays for reducing their pollutant effects and for their transformation into valuable products, the most suitable procedures being found to involve recycling rather than the detoxication of these wastes. Direct application of OMW to soil has been considered as an inexpensive method of disposal and recovery of their mineral and organic components but, because of their organic acid and phenol contents, OMW are also a source of pollution. By using composting technologies, it is possible to transform either fresh OMW or sludge from pond-stored OMW mixed with appropriate plant waste waterials (carriers) into organic fertilizers (composts) with no phytotoxicity to improve soil fertility and plant production, the process involving the microbial degradation of the polluting load of the wastes. Results of field and pot experiments using OMW-composts to cultivate horticultural and other crops have shown that yields obtained with organic fertilization are similar, and sometimes higher, to those obtained with a balanced mineral fertilizer. A comparison between the macro and micronutrient contents of plants cultivated with organic or mineral fertilizers did not generally reveal important differences. However, the cases of iron and manganese are worth mentioning as their bio-availability may be linked to the soil humic complexes originated by the OMW organic fertilizers.