Microbiome DNA Research Articles

Oral microbiota analyses of Saudi sickle cell anemics with dental caries

The objectives of this study were to identify the composition of oral microbiota in a cohort of patients with sickle cell anemia (SCA) and a high mean number of decayed, missing, and filled permanent teeth (DMFT) and compare it to a cohort of patients with SCA and a low number of DMFT and elucidate the effect of fetal haemoglobin levels on the oral microbiota composition. Patients who had been diagnosed with SCA, who were homozygous for sickling β-globin mutation (βS/βS), who had Arab-Indian haplotype, and who ranged in age from 5 to 12 years were included in this study. Oral saliva from each participant (n=100) was collected in GeneFiX™ Saliva DNA Microbiome Collection tube and DNA was extracted using GeneFiX™ DNA Isolation Kits. The composition of oral 16S rRNA from patients with SCA and high dental caries (n=27, DMFT ≥5) and low dental caries (n=73, DMFT ≤4) was analysed. Sequencing was performed on an Ion Personal Genome Machine using, Ion PGM Hi-Q view Sequencing 400-bp kit. We observed an overall increase in abundance of Proteobacteria, Chloroflexi, and Bacteroidetes in the high DMFT index group compared to those with a low DMFT index. In addition, there was an overall increased abundance of microbiota from Proteobacteria, Fusobacteria, Firmicutes, and Bacteroidetes in the patients with SCA with low fetal haemoglobin compared to those with high fetal haemoglobin (P < .05). Enterobacteriaceae species were the most significant abundant species of bacteria found in both the high DMFT index group and low fetal haemoglobin cohort (P < .05). Our data indicate that SCA in Saudi patients with high DMFT have a higher predominance of pathogenic bacteria compared to those with low DMFT. Furthermore, SCA in Saudi patients with low fetal haemoglobin have a higher predominance of pathogenic bacteria compared to those with higher fetal haemoglobin.

Synbiotic Supplementation Modulates Gut Microbiota, Regulates β-Catenin Expression and Prevents Weight Gain in ob/ob Mice: Preliminary Findings.

Background: Obesity is one of the main health problems in the world today, and dysbiosis seems to be one of the factors involved. The aim of this study was to examine the impact of synbiotic supplementation on obesity and the microbiota in ob/ob mice. Twenty animals were divided into four groups: obese treated (OT), obese control (OC), lean treated (LT) and lean control (LC). All animals received a standard diet for 8 weeks. The treated groups received a synbiotic (Simbioflora-Invictus Farmanutrição Ltd., Sao Paulo, Brazil) in water, while the nontreated groups received only water. After 8 weeks, all animals were sacrificed, and gut tissue and stool samples were collected for mRNA isolation and microbiota analysis, respectively. β-Catenin, occludin, cadherin and zonulin in the gut tissue were analyzed via RT-qPCR. Microbiome DNA was extracted from stool samples and sequenced using an Ion PGM Torrent platform. Results: Synbiotic supplementation reduced body weight gain in the OT group compared with the OC group (p = 0.0398) and was associated with an increase in Enterobacteriaceae (p = 0.005) and a decrease in Cyanobacteria (p = 0.047), Clostridiaceae (p = 0.026), Turicibacterales (p = 0.005) and Coprococcus (p = 0.047). On the other hand, a significant reduction in Sutterella (p = 0.009) and Turicibacter (p = 0.005) bacteria was observed in the LT group compared to the LC group. Alpha and beta diversities were different among all treated groups. β-Catenin gene expression was significantly decreased in the gut tissue of the OT group (p ≤ 0.0001) compared to the other groups. No changes were observed in occludin, cadherin or zonulin gene expression in the gut tissue. Conclusions: Synbiotic supplementation prevents excessive weight gain, modulates the gut microbiota, and reduces β-catenin expression in ob/ob mice.

Developing whole cell standards for the microbiome field

BackgroundEffective standardisation of the microbiome field is essential to facilitate global translational research and increase the reproducibility of microbiome studies. In this study, we describe the development and validation of a whole cell reference reagent specific to the gut microbiome by the UK National Institute for Biological Standards and Control. We also provide and test a two-step reporting framework to allow microbiome researchers to quickly and accurately validate choices of DNA extraction, sequencing, and bioinformatic pipelines.ResultsUsing 20 strains that are commonly found in the gut, we developed a whole cell reference reagent (WC-Gut RR) for the evaluation of the DNA extraction protocols commonly used in microbiome pipelines. DNA was first analysed using the physicochemical measures of yield, integrity, and purity, which demonstrated kits widely differed in the quality of the DNA they produced. Importantly, the combination of the WC-Gut RR and the three physicochemical measures allowed us to differentiate clearly between kit performance. We next assessed the ability of WC-Gut RR to evaluate kit performance in the reconstitution of accurate taxonomic profiles. We applied a four-measure framework consisting of Sensitivity, false-positive relative abundance (FPRA), Diversity, and Similarity as previously described for DNA reagents. Using the WC-Gut RR and these four measures, we could reliably identify the DNA extraction kits’ biases when using with both 16S rRNA sequencing and shotgun sequencing. Moreover, when combining this with complementary DNA standards, we could estimate the relative bias contributions of DNA extraction kits vs bioinformatic analysis. Finally, we assessed WC-Gut RR alongside other commercially available reagents. The analysis here clearly demonstrates that reagents of lower complexity, not composed of anaerobic and hard-to-lyse strains from the gut, can artificially inflate the performance of microbiome DNA extraction kits and bioinformatic pipelines.ConclusionsWe produced a complex whole cell reagent that is specific for the gut microbiome and can be used to evaluate and benchmark DNA extractions in microbiome studies. Used alongside a DNA standard, the NIBSC DNA-Gut-Mix RR helps estimating where biases occur in microbiome pipelines. In the future, we aim to establish minimum thresholds for data quality through an interlaboratory collaborative study.BEU9jL7xnwncT84Rof_ZoXVideo

Reducing human DNA bias in cystic fibrosis airway specimens for microbiome analysis

Next generation sequencing (NGS) has transformed our understanding of airway microbiology, however there are methodology limitations that require consideration. The presence of high concentrations of human DNA in clinical specimens can significantly impact sequencing of the microbiome, especially in low biomass samples. Here we compared three different methods (0.025% saponin, NEBNext Microbiome DNA enrichment kit, QIAamp DNA microbiome kit) for the reduction of human DNA from six CF sputum samples and determined the impact on the microbiome detected using 16S rRNA gene sequencing.Human DNA in undepleted CF sputum accounted for 94.3% of the total DNA. Saponin, the NEBNext kit and the QIAamp kit reduced human DNA levels by an average of 38.7%, 61.8% and 94.8%, respectively. None of the depletion methods reduced total bacterial DNA concentrations. QIAamp depletion did not influence taxa richness or alpha diversity however alterations to the core genera were noted following depletion.While all methods reduced human DNA in the CF sputum samples, the QIAamp DNA microbiome kit reduced Human DNA levels significantly while leaving bacterial DNA levels unchanged. Human DNA depletion in low biomass, human DNA-dense CF sputum samples is vital for improving bacterial resolution in the CF airway microbiome.

Are short-read amplicons suitable for the prediction of microbiome functional potential? A critical perspective.

Taxonomic marker gene analysis allows uncovering taxonomic profiles of microbial communities at low cost, making it omnipresent in microbiome research. There is an ever-expanding set of tools to extract further biological information from this kind of data. In this perspective, we enunciate several concerns regarding the biological validity of predicting functional potential from taxonomic profiles, especially when they are generated by short-read sequencing. The taxonomic resolution of marker genes, intragenomic variability of marker genes, and the compositional nature of microbiome data are discussed. Combining actual measurements of microbiome functions with predicted functional potentials is proposed as a powerful approach to better understand microbiome functioning. In this context, the significance of predicted functional potentials for generating and testing hypotheses is highlighted. We argue that functions of microbiomes predicted from microbiome DNA read count data generated by short-read amplicon sequencing should not serve as the only basis to draw biological inferences.

Peripheral Blood Microbiome Analysis via Noninvasive Prenatal Testing Reveals the Complexity of Circulating Microbial Cell-Free DNA.

ABSTRACTWhile circulating cell-free DNA (cfDNA) is becoming a powerful marker for noninvasive identification of infectious pathogens in liquid biopsy specimens, a microbial cfDNA baseline in healthy individuals is urgently needed for the proper interpretation of microbial cfDNA sequencing results in clinical metagenomics. Because noninvasive prenatal testing (NIPT) shares many similarities with the sequencing protocol of metagenomics, we utilized the standard low-pass whole-genome-sequencing-based NIPT to establish a microbial cfDNA baseline in healthy people. Sequencing data from a total of 107,763 peripheral blood samples of healthy pregnant women undergoing NIPT screening were retrospectively collected and reanalyzed for microbiome DNA screening. It was found that more than 95% of exogenous cfDNA was from bacteria, 3% from eukaryotes, and 0.4% from viruses, indicating the gut/environment origins of many microorganisms. Overall and regional abundance patterns were well illustrated, with huge regional diversity and complexity, and unique interspecies and symbiotic relationships were observed for TORCH organisms (Toxoplasma gondii, others [Treponema pallidum {causing syphilis}, hepatitis B virus {HBV}, and human parvovirus B19 {HPV-B19}], rubella virus, cytomegalovirus [CMV], and herpes simplex virus [HSV]) and another common virus, Epstein-Barr virus (EBV). To sum up, our study revealed the complexity of the baseline circulating microbial cfDNA and showed that microbial cfDNA sequencing results need to be interpreted in a more comprehensive manner.IMPORTANCE While circulating cell-free DNA (cfDNA) has been becoming a powerful marker for noninvasive identification of infectious pathogens in liquid biopsy specimens, a baseline for microbial cfDNA in healthy individuals is urgently needed for the proper interpretation of microbial cfDNA sequencing results in clinical metagenomics. Standard low-pass whole-genome-sequencing-based NIPT shares many similarities with the sequencing protocol for metagenomics and could provide a microbial cfDNA baseline in healthy people; thus, a reference cfDNA data set of the human microbiome was established with sequencing data from a total of 107,763 peripheral blood samples of healthy pregnant women undergoing NIPT screening. Our study revealed the complexity of circulating microbial cfDNA and indicated that microbial cfDNA sequencing results need to be interpreted in a more comprehensive manner, especially with regard to geographic patterns and coexistence networks.

The Pre-Analytical CEN/TS Standard for Microbiome Diagnostics-How Can Research and Development Benefit?

Recently, CEN/TS 17626:2021, the European pre-analytical standard for human specimens intended for microbiome DNA analysis, was published. Although this standard relates to diagnostic procedures for microbiome analysis and is relevant for in vitro diagnostic (IVD) manufacturers and diagnostic laboratories, it also has implications for research and development (R&D). We present here why standards are needed in biomedical research, what pre-analytical standards can accomplish, and which elements of the pre-analytical workflow they cover. The benefits of standardization for the generation of FAIR (findable, accessible, interoperable, reusable) data and to support innovation are briefly discussed.

Selective Whole-Genome Amplification as a Tool to Enrich Specimens with Low Treponema pallidum Genomic DNA Copies for Whole-Genome Sequencing.

ABSTRACTDownstream next-generation sequencing (NGS) of the syphilis spirochete Treponema pallidum subspecies pallidum (T. pallidum) is hindered by low bacterial loads and the overwhelming presence of background metagenomic DNA in clinical specimens. In this study, we investigated selective whole-genome amplification (SWGA) utilizing multiple displacement amplification (MDA) in conjunction with custom oligonucleotides with an increased specificity for the T. pallidum genome and the capture and removal of 5′-C-phosphate-G-3′ (CpG) methylated host DNA using the NEBNext Microbiome DNA enrichment kit followed by MDA with the REPLI-g single cell kit as enrichment methods to improve the yields of T. pallidum DNA in isolates and lesion specimens from syphilis patients. Sequencing was performed using the Illumina MiSeq v2 500 cycle or NovaSeq 6000 SP platform. These two enrichment methods led to 93 to 98% genome coverage at 5 reads/site in 5 clinical specimens from the United States and rabbit-propagated isolates, containing >14 T. pallidum genomic copies/μL of sample for SWGA and >129 genomic copies/μL for CpG methylation capture with MDA. Variant analysis using sequencing data derived from SWGA-enriched specimens showed that all 5 clinical strains had the A2058G mutation associated with azithromycin resistance. SWGA is a robust method that allows direct whole-genome sequencing (WGS) of specimens containing very low numbers of T. pallidum, which has been challenging until now.IMPORTANCE Syphilis is a sexually transmitted, disseminated acute and chronic infection caused by the bacterial pathogen Treponema pallidum subspecies pallidum. Primary syphilis typically presents as single or multiple mucocutaneous lesions and, if left untreated, can progress through multiple stages with various clinical manifestations. Molecular studies often rely on direct amplification of DNA sequences from clinical specimens; however, this can be impacted by inadequate samples due to disease progression or timing of patients seeking clinical care. While genotyping has provided important data on circulating strains over the past 2 decades, WGS data are needed to better understand strain diversity, perform evolutionary tracing, and monitor antimicrobial resistance markers. The significance of our research is the development of an SWGA DNA enrichment method that expands the range of clinical specimens that can be directly sequenced to include samples with low numbers of T. pallidum.

Effect of Cold Storage on the Viable and Total Bacterial Populations in Human Milk.

Expression and cold storage of human milk is a common practice. Current guidelines for cold storage of expressed milk do not take into account the impact on the milk microbiome. Here, we investigated the impact of cold storage on viable bacterial populations in human milk. Freshly expressed milk samples (n = 10) were collected and analysed immediately, stored at 4 °C for four days, −20 °C for 2.25 months and 6 months, and −80 °C for 6 months. Samples were analysed using propidium monoazide (PMA; a cell viability dye) coupled with full-length 16S rRNA gene. An aliquot of each sample was additionally analysed without PMA to assess the impact of cold storage on the total DNA profile of human milk. Cold storage significantly altered the composition of both the viable microbiome and total bacterial DNA profile, with differences in the relative abundance of several OTUs observed across each storage condition. However, cold storage did not affect the richness nor diversity of the samples (PERMANOVA all p > 0.2). Storage of human milk under typical and recommended conditions results in alterations to the profile of viable bacteria, with potential implications for infant gut colonisation and infant health.

Structural Analysis of Salivary Bacterial and Fungal Microbiome in Oral Lichen Planus Patients

To explore the differences of oral mycobiome and bacteriome between the healthy controls (H) and oral lichen planus (OLP) patients, and the co-occurrence patterns of the salivary mycobiome and bacteriome and the association with host immunity. Saliva samples were collected from clinical OLP patients (n=35) and healthy volunteers (n=18). Microbiome DNA was extracted for bacterial 16S rRNA genes sequencing and fungal internal transcribed spacer 2 (ITS2) sequencing. Bioinformatics analysis was performed on the data.The levels of IL-17 and IL-23, two pro-inflammatory cytokines, in the saliva were examined, and their correlation with the bacteria was analyzed. There was no significant difference in the overall community structure of the mycobiome and the bacteriome between OLP patients and healthy controls. The abundance of Prevotellaand Solobacterium in the saliva bacteriome was significantly increased in the OLP group (P<0.05), and the relative abundance of Candidaand Aspergillusin the saliva mycobiome was also significantly increased (P<0.05). The co-occurrence pattern of the salivary mycobiome and bacteriome showed that the aforementioned difference was not related. However, the correlation between Aspergillusand bacteria was altered in the H group and the OLP group, and co-occurrence was reduced in the latter group. The level of IL-17 in the saliva was significantly increased in the OLP group. IL-17 and clinical scores were significantly correlated with the abundance of Porphyromonas. The increased abundance of Prevotella, Solobacterium, Candida, and Aspergillus was associated with the pathogenesis of OLP, and the changes of the microbiome co-occurrence relationship and host immunity may be involved in the pathogenesis of OLP.

Analyzing Large Microbiome Datasets Using Machine Learning and Big Data

Metagenomics promises to provide new valuable insights into the role of microbiomes in eukaryotic hosts such as humans. Due to the decreasing costs for sequencing, public and private repositories for human metagenomic datasets are growing fast. Metagenomic datasets can contain terabytes of raw data, which is a challenge for data processing but also an opportunity for advanced machine learning methods like deep learning that require large datasets. However, in contrast to classical machine learning algorithms, the use of deep learning in metagenomics is still an exception. Regardless of the algorithms used, they are usually not applied to raw data but require several preprocessing steps. Performing this preprocessing and the actual analysis in an automated, reproducible, and scalable way is another challenge. This and other challenges can be addressed by adjusting known big data methods and architectures to the needs of microbiome analysis and DNA sequence processing. A conceptual architecture for the use of machine learning and big data on metagenomic data sets was recently presented and initially validated to analyze the rumen microbiome. The same architecture can be used for clinical purposes as is discussed in this paper.

Circulating microbiome DNA: An emerging paradigm for cancer liquid biopsy.

Dysbiosis of the human microbiome has long been reported to be closely associated with various cancers. Accumulating studies have shown that microbial dysbiosis can accelerate tumorigenesis through tumor-promoting inflammation, DNA damage, and inducing immune evasion. Differential composition of microbiome could be novel biomarkers for cancer detection or biomarkers of successful immunotherapy. More importantly, emerging evidence demonstrates that alterations of circulating microbiome DNA (cmDNA) could serve as promising noninvasive biomarkers for cancer detection. It has been reported that distinct circulating bacterial DNA could distinguish prostate cancer, lung cancer, and melanoma patients from healthy populations. Therefore, in this review, we summarized current literature on microbial biomarkers for cancer detection and unraveled the potential of cmDNA as a promising cancer detection tool.

"More Guts Than Brains?"-The Role of Gut Microbiota in Idiopathic Intracranial Hypertension.

Idiopathic intracranial hypertension syndrome (IIH) is most common among obese women. Weight loss is an important factor in improving papilledema. Over the last decade, growing evidence has identified gut microbiota as a potential factor in the pathophysiology of obesity. Accordingly, we investigated whether the gut microbiome is modified in IIH patients compared with healthy controls, and provide possible new treatment venues. Shotgun metagenomic sequencing of the gut microbiome of 25 cases of IIH patients (according to the modified Dandy criteria) and 20 healthy controls. Participants were further stratified according to their body mass index. The total DNA from each sample was extracted using the PureLink Microbiome DNA Purification Kit A29789 (Invitrogen, Thermo Fisher Scientific, US). Library preparation was performed using the Nextera DNA Flex Library Prep Kit. Samples were sequenced on the Illumina Novaseq 6000 device. A list of bacterial species that significantly differed between the IIH patients and healthy controls was produced in addition to species diversity. In addition, patients' cohort alone was analyzed, (excluding the healthy controls), and the effect of acetazolamide treatment on their gut microbiota was analyzed. IIH patients have a lower diversity of bacterial species compared with healthy individuals. These bacteria, that is, Lactobacillus ruminis (L. ruminis) (p<6.95E-08), Atopobium parvulum (p<3.9E-03), Megamonas hypermegale (p<5.61E-03), Ruminococcus gnavus (p<1.29E-02), MEL.A1 (p<3.04E-02), and Streptococcus sp. I-G2 (p<3.04E-02), were previously characterized with beneficial health effects. Moreover, we found that Lactobacillus brevis, a beneficial bacterium as well, is more abundant in acetazolamide treated patients (p<7.07E-06). Gut microbiota plays a potential role in IIH etiology and therefore, can provide a promising new treatment approach for this disease.

Depressive hypertension: A proposed human endotype of brain/gut microbiome dysbiosis

Hypertension (HTN) is frequently linked with depression (DEP) in adults with cardiovascular disease (CVD), yet the underlying mechanism and successful management remain elusive. We approached this knowledge gap through the lens that humans are eukaryote-prokaryote "meta-organisms," such that cardiovascular disease dysregulation is a mosaic disorder involving dysbiosis of the gut. We hypothesized that patients diagnosed with hypertension plus depression harbor a unique gut microbial ecology with attending functional genomics engaged with their hosts' gut/brain axis physiology. Stool microbiome DNA was analyzed by whole metagenome shotgun sequencing in 54 subjects parsed into cohorts diagnosed with HTN only (N = 18), DEP only (N = 7), DEP plus HTN (DEP-HTN) (N = 8), or reference subjects with neither HTN nor DEP (N = 21). A novel battery of machine-learning multivariate analyses of de-noised data yielded effect sizes and permutational covariance-based dissimilarities that significantly differentiated the cohorts (false discovery rate (FDR)-adjusted P ≤ .05); data clustering within 95% confidence interval). Metagenomic significant differences extricated the four cohorts. Data of the cohort exhibiting DEP-HTN were germane to the interplay of central control of blood pressure concomitant with the neuropathology of depressive disorders. DEP-HTN gut bacterial community ecology was defined by co-occurrence of Eubacterium siraeum, Alistipes obesi, Holdemania filiformis, and Lachnospiraceae bacterium 1.1.57FAA with Streptococcus salivariu. The corresponding microbial functional genomics of DEP-HTN engaged pathways degrading GABA and beneficial short chain fatty acids (SCFA), and are associated with enhanced sodium absorption and inflammasome induction. These data suggest a new putative endotype of hypertension, which we denote "depressive-hypertension" (DEP-HTN), for which we posit a model that is distinctive from either HTN alone or DEP alone. An "endotype" is a subtype of a heterogeneous pathophysiological mechanism. The DEP-HTN model incorporates a unique signature of microbial taxa and functional genomics with crosstalk that putatively intertwines host pathophysiology involving the gastrointestinal tract with disruptions in central control of blood pressure and mood. The DEP-HTN endotype model engages cardiology with gastroenterology and psychiatry, providing a proof-of-concept foundation to explore future treatments, diagnosis, and prevention of HTN-coupled mood disorders.

Wykorzystanie potencjału mikrobiomu środowiskowego w kryminalistyce

Biometrics is one of the basic detection techniques used in law enforcement activities on a daily basis. Like other techniques, it is constantly changing. This article aims to provide an overview of what was in the past, what is now, and what will be in the near future. It presents the selected methods of collecting some of the data, as well as the systems used to rocess them. In addition, practical guidance is provided on how individual biometric data should be collected and, for specific cases, it explains why the data collection procedure is carried out in one way and not another, and what it means for its subsequent processing. As problems sometimes arise during recovery of the material they have been presented together with an overview of the reasons for that. In addition to information on the technical aspects, the article also includes references to legal acts regulatingissues related to biometrics.

Dysbiotic Lesional Microbiome With Filaggrin Missense Variants Associate With Atopic Dermatitis in India.

Background: Atopic Dermatitis (AD) has been associated with the loss of function (LoF) mutations in Filaggrin (FLG) gene and increase in relative abundance of specific microbes in the lesional skin, predominantly in Caucasians. Our study aims to determine, in Indian AD patients, (a) the prevalence of FLG LoF and missense mutations, and (b) the nature and extent of dysbiosis and altered microbial pathways with and without mutations in FLG. AD patients (n = 34) and healthy controls (n = 54) were recruited from India in this study and shotgun sequencing was carried out in a subset of samples with adequate microbiome DNA concentration. Host DNA from the same subset of samples was subjected to FLG coding region sequencing and host-microbiome association was estimated.Results: The prevalence of FLG LoFs that are associated with AD globally were significantly lesser in our cases and controls (8.6%, 0%) than those reported in Europeans (27%, 2.6%). Staphylococcus aureus was present only on AD skin [abundance in Pediatric AD: 32.86%; Adult AD: 22.17%], but not on healthy skin on which Staphylococcus hominis (Adult controls: 16.43%, Adult AD: 0.20%; p = 0.002), Cutibacterium acnes (Adult controls:10.84%, Adult AD: 0.90%; p = 0.02), and Malassezia globosa (Adult controls: 8.89%, Adult AD: 0.005%; p = 0.001) were significantly more abundant. Microbial pathways mostly associated with skin barrier permeability, ammonia production and inflammation (Arginine and Proline metabolism, Histidine Metabolism and Staphylococcus aureus infection) were significantly enriched on AD skin metagenome. These pathways are also reported to impair antimicrobial peptide activity. Among AD patients with missense single nucleotide polymorphisms harboring “potentially damaging” alleles in FLG gene, damaging allele dosage was significantly (p < 0.02) positively correlated with relative abundance of phylum_Proteobacteria up to order_Pseudomonadales and negatively correlated with phylum_Firmicutes up to species_Staphylococcus aureus.Conclusion: Our study has provided evidence that host DNA profile is significantly associated with microbiome composition in the development of AD. Species and strain level analysis showed that the microbial pathways enriched in AD cases were mostly found in MRSA strains. These evidences can be harnessed to control AD by modulating the microbiome using a personalized strategy. Our findings on the association of FLG genotypes with the microbiome dysbiosis may pave the way for a personalized strategy to provide a more effective control of AD.

Proteome and Microbiome Mapping of Human Gingival Tissue in Health and Disease.

Efforts to map gingival tissue proteomes and microbiomes have been hampered by lack of sufficient tissue extraction methods. The pressure cycling technology (PCT) is an emerging platform for reproducible tissue homogenisation and improved sequence retrieval coverage. Therefore, we employed PCT to characterise the proteome and microbiome profiles in healthy and diseased gingival tissue. Healthy and diseased contralateral gingival tissue samples (total n = 10) were collected from five systemically healthy individuals (51.6 ± 4.3 years) with generalised chronic periodontitis. The tissues were then lysed and digested using a Barocycler, proteins were prepared and submitted for mass spectrometric analysis and microbiome DNA for 16S rRNA profiling analysis. Overall, 1,366 human proteins were quantified (false discovery rate 0.22%), of which 69 proteins were differentially expressed (≥2 peptides and p < 0.05, 62 up, 7 down) in periodontally diseased sites, compared to healthy sites. These were primarily extracellular or vesicle-associated proteins, with functions in molecular transport. On the microbiome level, 362 species-level operational taxonomic units were identified. Of those, 14 predominant species accounted for >80% of the total relative abundance, whereas 11 proved to be significantly different between healthy and diseased sites. Among them, Treponema sp. HMT253 and Fusobacterium naviforme and were associated with disease sites and strongly interacted (r > 0.7) with 30 and 6 up-regulated proteins, respectively. Healthy-site associated strains Streptococcus vestibularis, Veillonella dispar, Selenomonas sp. HMT478 and Leptotrichia sp. HMT417 showed strong negative interactions (r < −0.7) with 31, 21, 9, and 18 up-regulated proteins, respectively. In contrast the down-regulated proteins did not show strong interactions with the regulated bacteria. The present study identified the proteomic and intra-tissue microbiome profile of human gingiva by employing a PCT-assisted workflow. This is the first report demonstrating the feasibility to analyse full proteome profiles of gingival tissues in both healthy and disease sites, while deciphering the tissue site-specific microbiome signatures.

Complementary sampling methods for coral histology, metabolomics and microbiome

Abstract Underwater visual surveys of coral reefs are the primary method managers use to monitor coral health. However, these surveys are limited to visual signs, such as bleaching and tissue loss lesions, which occur only after significant stress has accumulated. More holistic characterization of coral health can allow for better monitoring of reef changes across natural environmental gradients, in response to anthropogenic stress and after disturbance events (e.g. disease outbreaks, mass bleaching, dredging, run‐off events). Various methods exist to evaluate the health of the coral holobiont that do not depend on visual signs, including histological assessment, microbiome dysbiosis and metabolic profiles, yet these tools are rarely deployed concurrently. We present a clear, readily deployable protocol for sampling and preserving coral fragments, including (a) extraction of coral metabolites for analysis, (b) preservation of microbiome DNA for sequencing and (c) preservation of coral tissues for histopathology. Combined with visual surveys, these methods provide an unparalleled, holistic characterization of coral health. We provide a field‐tested, optimized protocol for conducting coral sampling. This protocol guides the user through concurrent assessments of coral tissue structure and the holobiont microbiome and metabolome, and directs the user to useful resources for downstream data analysis. This protocol facilitates quantitative characterization of coral health beyond visual surveys alone, which is a valuable step forward in reef research and management and will improve our ability to describe, model and mediate impacts to coral reefs.

Culture-dependent to culture-independent approaches for the bioremediation of paints: a review

Environmental pollution by paint effluents is a public health threat, distressing total biodiversity and environment. Environmental treatment options like bioremediation mediated by microorganisms is still developing and is considered as a modern approach for the environmentalists worldwide. Thus present review was sought to explore the culture-dependent (isolation) to culture-independent (multi-omics) approaches for the bioremediation of paints. Bioremediation is once considered as safe, reliable and eco-friendly yet facing challenges. Microbes exhibiting bioremediation potential were screened and characterized but, in most of the studies, cannot completely utilize the targeted pollutant or were unsuccessful against pollutant at in situ settings. Thereafter, consortia studies were conducted and found that microbial performance remained excellent in synergistic while negligible in antagonistic association. Consortia studies were most frequently carried out using bacteria while few studies were conducted on the fungi alone and their association with bacteria. Moreover, the combined strategies of consortia, genes and enzymes were also elucidated and later the genetically modified microbes were also tested for in situ cleanup. Nowadays, metagenomics approaches which reveal the functional gene information of microbes derived from the DNA of microbiome with different physiological states were studied for environmental cleanup. At present from the literature insight, it is predicted that the bioremediation future lies in the understanding of metaphenome (product of metagenome and available resources).

Multi-omic profiling reveals associations between the gut mucosal microbiome, the metabolome, and host DNA methylation associated gene expression in patients with colorectal cancer

BackgroundThe human gut microbiome plays a critical role in the carcinogenesis of colorectal cancer (CRC). However, a comprehensive analysis of the interaction between the host and microbiome is still lacking.ResultsWe found correlations between the change in abundance of microbial taxa, butyrate-related colonic metabolites, and methylation-associated host gene expression in colonic tumour mucosa tissues compared with the adjacent normal mucosa tissues. The increase of genus Fusobacterium abundance was correlated with a decrease in the level of 4-hydroxybutyric acid (4-HB) and expression of immune-related peptidase inhibitor 16 (PI16), Fc Receptor Like A (FCRLA) and Lymphocyte Specific Protein 1 (LSP1). The decrease in the abundance of another potentially 4-HB-associated genus, Prevotella 2, was also found to be correlated with the down-regulated expression of metallothionein 1 M (MT1M). Additionally, the increase of glutamic acid-related family Halomonadaceae was correlated with the decreased expression of reelin (RELN). The decreased abundance of genus Paeniclostridium and genus Enterococcus were correlated with increased lactic acid level, and were also linked to the expression change of Phospholipase C Beta 1 (PLCB1) and Immunoglobulin Superfamily Member 9 (IGSF9) respectively. Interestingly, 4-HB, glutamic acid and lactic acid are all butyrate precursors, which may modify gene expression by epigenetic regulation such as DNA methylation.ConclusionsOur study identified associations between previously reported CRC-related microbial taxa, butyrate-related metabolites and DNA methylation-associated gene expression in tumour and normal colonic mucosa tissues from CRC patients, which uncovered a possible mechanism of the role of microbiome in the carcinogenesis of CRC. In addition, these findings offer insight into potential new biomarkers, therapeutic and/or prevention strategies for CRC.