Design, Optimization, and Application of a 3D-Printed Polymer Sample Introduction System for the ICP-MS Analysis of Nanoparticles and Cells.

Commonly used sample introduction systems for inductively coupled plasma mass spectrometry (ICP-MS) are generally not well-suited for single particle ICP-MS (spICP-MS) applications due to their high sample requirements and low efficiency. In this study, the first completely 3D-printed, polymer SIS was developed to facilitate spICP-MS analysis. The system is based on a microconcentric pneumatic nebulizer and a single-pass spray chamber with an additional sheath gas flow to further facilitate the transport of larger droplets or particles. The geometry of the system was optimized using numerical simulations. Its aerosol characteristics and operational conditions were studied via optical particle counting and a course of spICP-MS measurements, involving nanodispersions and cell suspensions. In a comparison of the performance of the new and the standard (quartz microconcentric nebulizer plus a double-pass spray chamber) systems, it was found that the new sample introduction system has four times higher particle detection efficiency, significantly better signal-to-noise ratio, provides ca. 20% lower size detection limit, and allows an extension of the upper limit of transportable particle diameters to about 25 µm.

Dual RNA-Seq Profiling Unveils Mycoparasitic Activities of Trichoderma atroviride against Haploid Armillaria ostoyae in Antagonistic Interaction Assays.

Armillaria ostoyae, a species among the destructive forest pathogens from the genus Armillaria, causes root rot disease on woody plants worldwide. Efficient control measures to limit the growth and impact of this severe underground pathogen are under investigation. In a previous study, a new soilborne fungal isolate, Trichoderma atroviride SZMC 24276 (TA), exhibited high antagonistic efficacy, which suggested that it could be utilized as a biocontrol agent. The dual culture assay results indicated that the haploid A. ostoyae-derivative SZMC 23085 (AO) (C18/9) is highly susceptible to the mycelial invasion of TA. In the present study, we analyzed the transcriptome of AO and that of TA in in vitro dual culture assays to test the molecular arsenal of Trichoderma antagonism and the defense mechanisms of Armillaria. We conducted time-course analysis and functional annotation and analyzed enriched pathways and differentially expressed genes including biocontrol-related candidate genes from TA and defense-related candidate genes from AO. The results indicated that TA deployed several biocontrol mechanisms when confronted with AO. In response, AO initiated multiple defense mechanisms to protect against the fungal attack. To our knowledge, the present study offers the first transcriptome analysis of a biocontrol fungus attacking AO. Overall, this study provides insights that aid the further exploration of plant pathogen-biocontrol agent interaction mechanisms. IMPORTANCE Armillaria species can survive for decades in the soil on dead woody debris, develop rapidly under favorable conditions, and harmfully infect newly planted forests. Our previous study found Trichoderma atroviride to be highly effective in controlling Armillaria growth; therefore, our current work explored the molecular mechanisms that might play a key role in Trichoderma-Armillaria interactions. Direct confrontation assays combined with time course-based dual transcriptome analysis provided a reliable system for uncovering the interactive molecular dynamics between the fungal plant pathogen and its mycoparasitic partner. Furthermore, using a haploid Armillaria isolate allowed us to survey the deadly prey-invading activities of the mycoparasite and the ultimate defensive strategies of its prey. Our current study provides detailed insights into the essential genes and mechanisms involved in Armillaria defense against Trichoderma and the genes potentially involved in the efficiency of Trichoderma to control Armillaria. In addition, using a sensitive haploid Armillaria strain (C18/9), with its complete genome data already available, also offers the opportunity to test possible variable molecular responses of Armillaria ostoyae toward diverse Trichoderma isolates with various biocontrol abilities. Initial molecular tests of the dual interactions may soon help to develop a targeted biocontrol intervention with mycoparasites against plant pathogens.

Microbiomes in supragingival biofilms and saliva of adolescents with gingivitis and gingival health.

BACKGROUND: Important alterations exist in the microbiomes of supragingival biofilm and saliva samples from adolescent patients developing induced or spontaneous gingivitis relative to healthy controls. These and the relationships to dental health are not fully understood yet. SUBJECTS AND METHODS: Supragingival biofilm samples (n = 36) were collected from the teeth of 9 adolescents with gingivitis induced by orthodontic appliances, as well as dental plaques (n = 40) from 10 adolescents with spontaneous gingivitis, in addition to similar samples (n = 36) from 9 healthy controls. The bacterial metagenomes were analyzed by 16S rRNA gene amplicon sequencing. Salivary microbiomes of the same persons were characterized by shotgun metagenome sequencing. The data sets were examined using advanced bioinformatics workflows and two reference databases. RESULTS: The composition and diversity of bacterial communities did not differ extensively among the three study groups. Nevertheless, the relative abundances of the genera Fusobacterium, Akkermansia, Treponema, and Campylobacter were prominently higher in gingivitis patients versus controls. In contrast, the genera Lautropia, Kingella, Neisseria, Actinomyces, and Rothia were significantly more abundant in controls than in either of the two gingivitis groups. CONCLUSIONS: The abundance pattern of certain taxa rather than individual strains shows characteristic features of potential diagnostic value. Stringent bioinformatics treatment of the sequencing data is mandatory to avoid unintentional misinterpretations.

Genome-centric investigation of anaerobic digestion using sustainable second and third generation substrates.

Biogas production through co-digestion of second and third generation substrates is an environmentally sustainable approach. Green willow biomass, chicken manure waste and microalgae biomass substrates were combined in the anaerobic digestion experiments. Biochemical methane potential test showed that biogas yields of co-digestions were significantly higher compared to the yield when energy willow was the sole substrate. To scale up the experiment continuous stirred-tank reactors (CSRTs) are employed, digestion parameters are monitored. Furthermore, genome-centric metagenomics approach was employed to gain functional insight into the complex anaerobic decomposing process. This revealed the importance of Firmicutes, Actinobacteria, Proteobacteria and Bacteroidetes phyla as major bacterial participants, while Methanomicrobia and Methanobacteria represented the archaeal constituents of the communities. The bacterial phyla were shown to perform the carbohydrate hydrolysis. Among the representatives of long-chain carbohydrate hydrolysing microbes Bin_61: Clostridia is newly identified metagenome assembled genome (MAG) and Bin_13: DTU010 sp900018335 is common and abundant in all CSTRs. Methanogenesis was linked to the slow-growing members of the community, where hydrogenotrophic methanogen species Methanoculleus (Bin_10) and Methanobacterium (Bin_4) predominate. A sensitive balance between H2 producers and consumers was shown to be critical for stable biomethane production and efficient waste biodegradation.

Toward Personalized Oral Diagnosis: Distinct Microbiome Clusters in Periodontitis Biofilms.

Periodontitis is caused by pathogenic subgingival microbial biofilm development and dysbiotic interactions between host and hosted microbes. A thorough characterization of the subgingival biofilms by deep amplicon sequencing of 121 individual periodontitis pockets of nine patients and whole metagenomic analysis of the saliva microbial community of the same subjects were carried out. Two biofilm sampling methods yielded similar microbial compositions. Taxonomic mapping of all biofilms revealed three distinct microbial clusters. Two clinical diagnostic parameters, probing pocket depth (PPD) and clinical attachment level (CAL), correlated with the cluster mapping. The dysbiotic microbiomes were less diverse than the apparently healthy ones of the same subjects. The most abundant periodontal pathogens were also present in the saliva, although in different representations. The single abundant species Tannerella forsythia was found in the diseased pockets in about 16-17-fold in excess relative to the clinically healthy sulcus, making it suitable as an indicator of periodontitis biofilms. The discrete microbial communities indicate strong selection by the host immune system and allow the design of targeted antibiotic treatment selective against the main periodontal pathogen(s) in the individual patients.

Chlorella vulgaris and Its Phycosphere in Wastewater: Microalgae-Bacteria Interactions During Nutrient Removal.

Microalgae-based bioenergy production is a promising field with regard to the wide variety of algal species and metabolic potential. The use of liquid wastes as nutrient clearly improves the sustainability of microalgal biofuel production. Microalgae and bacteria have an ecological inter-kingdom relationship. This microenvironment called phycosphere has a major role in the ecosystem productivity and can be utilized both in bioremediation and biomass production. However, knowledge on the effects of indigenous bacteria on microalgal growth and the characteristics of bacterial communities associated with microalgae are limited. In this study municipal, industrial and agricultural liquid waste derivatives were used as cultivation media. Chlorella vulgaris green microalgae and its bacterial partners efficiently metabolized the carbon, nitrogen and phosphorous content available in these wastes. The read-based metagenomics approach revealed a diverse microbial composition at the start point of cultivations in the different types of liquid wastes. The relative abundance of the observed taxa significantly changed over the cultivation period. The genome-centric reconstruction of phycospheric bacteria further explained the observed correlations between the taxonomic composition and biomass yield of the various waste-based biodegradation systems. Functional profile investigation of the reconstructed microbes revealed a variety of relevant biological processes like organic acid oxidation and vitamin B synthesis. Thus, liquid wastes were shown to serve as valuable resources of nutrients as well as of growth promoting bacteria enabling increased microalgal biomass production.

A case study of salivary microbiome in smokers and non-smokers in Hungary: analysis by shotgun metagenome sequencing.

OBJECTIVE: To investigate the role of cigarette smoking in disease-development through altering the composition of the oral microbial community. Periodontitis and oral cancer are highly prevalent in Hungary; therefore, the salivary microbiome of smoker and non-smoker Hungarian adults was characterized. METHODS: Shotgun metagenome sequencing of salivary DNA samples from 22 individuals (11 non-smokers and 11 current smokers) was performed using the Ion Torrent PGMTM platform. Quality-filtered reads were analysed by both alignment-based sequence similarity searches and genome-centric binning. RESULTS: Prevotella, Veillonella and Streptococcus were the predominant genera in the saliva of both groups. Although the overall composition and diversity of the microbiota were similar, Prevotella was significantly more abundant in salivary samples of current smokers compared to non-smokers. Members of the genus Prevotella were implicated in the development of inflammatory diseases and oral cancer. The abundance of the genus Megasphaera also increased in current smokers, whereas the genera Neisseria, Oribacterium, Capnocytophaga and Porphyromonas were significantly reduced. The data generated by read-based taxonomic classification and genome-centric binning mutually validated the two distinct metagenomic approaches. CONCLUSION: Smoking-associated dysbiosis of the salivary microbiome in current cigarette smokers, especially increased abundance of Prevotella and Megasphaera genera, may facilitate disease development.

Integrated evolutionary analysis reveals antimicrobial peptides with limited resistance.

Antimicrobial peptides (AMPs) are promising antimicrobials, however, the potential of bacterial resistance is a major concern. Here we systematically study the evolution of resistance to 14 chemically diverse AMPs and 12 antibiotics in Escherichia coli. Our work indicates that evolution of resistance against certain AMPs, such as tachyplesin II and cecropin P1, is limited. Resistance level provided by point mutations and gene amplification is very low and antibiotic-resistant bacteria display no cross-resistance to these AMPs. Moreover, genomic fragments derived from a wide range of soil bacteria confer no detectable resistance against these AMPs when introduced into native host bacteria on plasmids. We have found that simple physicochemical features dictate bacterial propensity to evolve resistance against AMPs. Our work could serve as a promising source for the development of new AMP-based therapeutics less prone to resistance, a feature necessary to avoid any possible interference with our innate immune system.

Chicken or the Egg: Microbial Alterations in Biopsy Samples of Patients with Oral Potentially Malignant Disorders.

Oral carcinogenesis often leads to the alteration of the microbiota at the site of the tumor, but data are scarce regarding the microbial communities of oral potentially malignant disorders (OPMDs). Punch biopsies were taken from healthy and non-healthy mucosa of OPMD patients to analyze the microbiome using metagenome sequencing. In healthy oral mucosa biopsies the bacterial phyla Firmicutes, Fusobacteria, Proteobacteria, Actinobacteria and Bacteroidetes were detected by Ion Torrent sequencing. The same phyla as well as the phyla Fibrobacteres and Spirochaetes were present in the OPMD biopsies. On the species level, there were 10 bacterial species unique to the healthy tissue and 35 species unique to the OPMD lesions whereas eight species were detected in both samples. We observed that the relative abundance of Streptococcus mitis decreased in the OPMD lesions compared to the uninvolved tissue. In contrast, the relative abundance of Fusobacterium nucleatum, implicated in carcinogenesis, was elevated in OPMD. We detected markedly increased bacterial diversity in the OPMD lesions compared to the healthy oral mucosa. The ratio of S. mitis and F. nucleatum are characteristically altered in the OPMD lesions compared to the healthy mucosa.

Antimicrobial Activity of NCR Plant Peptides Strongly Depends on the Test Assays.

The symbiosis specific NCR247 and NCR335 cationic plant peptides of Medicago truncatula have been shown to exert antimicrobial activity against a wide range of microbes. However, their antimicrobial efficiency is clearly limited by divalent cations. Here, the antibacterial and antifungal activities of NCR247 and NCR335 peptides were compared to those of the well-characterized peptide antibiotics polymyxin B and the aminoglycoside streptomycin on three model microbes, Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae as representatives of Gram-negative and Gram-positive bacteria as well as eukaryotic fungi. The aim of the study was to assess how the killing efficiency of these peptides depends on various, widely used antimicrobial susceptibility assays. Validated resazurin microdilution assay was used to determine minimal growth inhibitory concentrations in three general test media (MHB, MHBII and low-salt medium LSM). Bactericidal/fungicidal activities were determined by the commonly used drop plate assay. The natural plant peptides showed distinct characteristics, NCR247 had a generally high sensitivity for Ca2+ and Mg2+ in the medium, while NCR335 proved to be a robust and strong antimicrobial agent with comparable efficiency values to polymyxin B. Activity data were confirmed visually, both NCR247 and NCR335 treatments at minimal bactericidal concentrations induced complete disruption of the membranes and provoked cell lysis on all tested microorganisms as observed by scanning electron microscopy.

Binary mixture of Satureja hortensis and Origanum vulgare subsp. hirtum essential oils: in vivo therapeutic efficiency against Helicobacter pylori infection.

BACKGROUND: Helicobacter pylori can cause many gastrointestinal and also extra-gastrointestinal disorders and is a major risk factor for gastric carcinoma and MALT lymphoma. Currently, numerous antibiotic-based therapies are available; however, these therapies have numerous drawbacks, mainly due to increasing prevalence of antibiotic resistant strains. Thus, there is an urgent need to develop novel therapeutic agents against H. pylori infections. MATERIALS AND METHODS: In this study, the anti-H. pylori activity of 2:1 mixture of Satureja hortensis and Origanum vulgare subsp. hirtum essential oils (2MIX) was investigated in vivo. After screening in vitro cytotoxicity of 2MIX on mammalian cell lines, the therapeutic efficiency was studied in a mouse model, where changes in H. pylori colonization were detected by PCR and histology of gastric samples. The immune reaction of mice was tested based on cytokine and chemokine production, and the in vivo toxicity of 2MIX was also investigated by measuring ALT and AST enzyme activities and Cyp3a11 and HO-1 mRNA levels in livers of mice. RESULTS: 2MIX had not shown in vitro cytotoxicity against cell lines, only the highest concentration caused significant decrease in their survival rates. In the in vivo experiments, 2MIX successfully eradicated the pathogen in 70% of the mice. We could not detect toxicity or altered cytokine and chemokine balance after in vivo treatments in mice. CONCLUSIONS: These results show that 2MIX is effective in reducing H. pylori colonization suggesting that this essential oil mixture has great potential as a new, effective, and safe therapeutic agent against H. pylori.

Surpassing the current limitations of biohydrogen production systems: The case for a novel hybrid approach.

The steadily increase of global energy requirements has brought about a general agreement on the need for novel renewable and environmentally friendly energy sources and carriers. Among the alternatives to a fossil fuel-based economy, hydrogen gas is considered a game-changer. Certain methods of hydrogen production can utilize various low-priced industrial and agricultural wastes as substrate, thus coupling organic waste treatment with renewable energy generation. Among these approaches, different biological strategies have been investigated and successfully implemented in laboratory-scale systems. Although promising, several key aspects need further investigation in order to push these technologies towards large-scale industrial implementation. Some of the major scientific and technical bottlenecks will be discussed, along with possible solutions, including a thorough exploration of novel research combining microbial dark fermentation and algal photoheterotrophic degradation systems, integrated with wastewater treatment and metabolic by-products usage.

Temperature-dependent transformation of biogas-producing microbial communities points to the increased importance of hydrogenotrophic methanogenesis under thermophilic operation.

Stability of biogas production is highly dependent on the microbial community composition of the bioreactors. This composition is basically determined by the nature of biomass substrate and the physical-chemical parameters of the anaerobic digestion. Operational temperature is a major factor in the determination of the anaerobic degradation process. Next-generation sequencing (NGS)-based metagenomic approach was used to monitor the organization and operation of the microbial community throughout an experiment where mesophilic reactors (37°C) were gradually switched to thermophilic (55°C) operation. Temperature adaptation resulted in a clearly thermophilic community having a generally decreased complexity compared to the mesophilic system. A temporary destabilization of the system was observed, indicating a lag phase in the community development in response to temperature stress. Increased role of hydrogenotrophic methanogens under thermophilic conditions was shown, as well as considerably elevated levels of Fe-hydrogenases and hydrogen producer bacteria were observed in the thermophilic system.

Revealing the factors influencing a fermentative biohydrogen production process using industrial wastewater as fermentation substrate.

BACKGROUND: Biohydrogen production through dark fermentation using organic waste as a substrate has gained increasing attention in recent years, mostly because of the economic advantages of coupling renewable, clean energy production with biological waste treatment. An ideal approach is the use of selected microbial inocula that are able to degrade complex organic substrates with simultaneous biohydrogen generation. Unfortunately, even with a specifically designed starting inoculum, there is still a number of parameters, mostly with regard to the fermentation conditions, that need to be improved in order to achieve a viable, large-scale, and technologically feasible solution. In this study, statistics-based factorial experimental design methods were applied to investigate the impact of various biological, physical, and chemical parameters, as well as the interactions between them on the biohydrogen production rates. RESULTS: By developing and applying a central composite experimental design strategy, the effects of the independent variables on biohydrogen production were determined. The initial pH value was shown to have the largest effect on the biohydrogen production process. High-throughput sequencing-based metagenomic assessments of microbial communities revealed a clear shift towards a Clostridium sp.-dominated environment, as the responses of the variables investigated were maximized towards the highest H2-producing potential. Mass spectrometry analysis suggested that the microbial consortium largely followed hydrogen-generating metabolic pathways, with the simultaneous degradation of complex organic compounds, and thus also performed a biological treatment of the beer brewing industry wastewater used as a fermentation substrate. CONCLUSIONS: Therefore, we have developed a complex optimization strategy for batch-mode biohydrogen production using a defined microbial consortium as the starting inoculum and beer brewery wastewater as the fermentation substrate. These results have the potential to bring us closer to an optimized, industrial-scale system which will serve the dual purpose of wastewater pre-treatment and concomitant biohydrogen production.

Monitoring of thermophilic adaptation of mesophilic anaerobe fermentation of sugar beet pressed pulp.

Anaerobe fermentation of sugar beet pressed pulp was investigated in pilot-scale digesters. Thermophilic adaptation of mesophilic culture was monitored using chemical analysis and metagenomic characterization of the sludge. Temperature adaptation was achieved by increasing the temperature gradually (2 °C day(-1)) and by greatly decreasing the OLR. During stable run, the OLR was increased gradually to 11.29 kg VS m(-3)d(-1) and biogas yield was 5% higher in the thermophilic reactor. VFA levels increased in the thermophilic reactor with increased OLR (acetic acid 646 mg L(-1), propionic acid 596 mg L(-1)), then VFA decreased and the operation was manageable beside the relative high tVFA (1300-2000 mg L(-1)). The effect of thermophilic adaptation on the microbial communities was studied using a sequencing-based metagenomic approach. Connections between physico-chemical parameters and populations of bacteria and methanogen archaea were revealed.

Antimicrobial nodule-specific cysteine-rich peptides induce membrane depolarization-associated changes in the transcriptome of Sinorhizobium meliloti.

Leguminous plants establish symbiosis with nitrogen-fixing alpha- and betaproteobacteria, collectively called rhizobia, which provide combined nitrogen to support plant growth. Members of the inverted repeat-lacking clade of legumes impose terminal differentiation on their endosymbiotic bacterium partners with the help of the nodule-specific cysteine-rich (NCR) peptide family composed of close to 600 members. Among the few tested NCR peptides, cationic ones had antirhizobial activity measured by reduction or elimination of the CFU and uptake of the membrane-impermeable dye propidium iodide. Here, the antimicrobial spectrum of two of these peptides, NCR247 and NCR335, was investigated, and their effect on the transcriptome of the natural target Sinorhizobium meliloti was characterized. Both peptides were able to kill quickly a wide range of Gram-negative and Gram-positive bacteria; however, their spectra were only partially overlapping, and differences were found also in their efficacy on given strains, indicating that the actions of NCR247 and NCR335 might be similar though not identical. Treatment of S. meliloti cultures with either peptide resulted in a quick downregulation of genes involved in basic cellular functions, such as transcription-translation and energy production, as well as upregulation of genes involved in stress and oxidative stress responses and membrane transport. Similar changes provoked mainly in Gram-positive bacteria by antimicrobial agents were coupled with the destruction of membrane potential, indicating that it might also be a common step in the bactericidal actions of NCR247 and NCR335.