Metataxonomic Approach Research Articles

Prokaryotic diversity in stream sediments affected by acid mine drainage.

The microbial communities in mining impacted areas rely on a variety of mechanisms to survive in such extreme environments. In this work, a meta-taxonomic approach using 16S rRNA gene sequences was used to investigate the prokaryotic diversity of sediment samples from water bodies affected by acid mine drainage at the São Domingos mining area in the south of Portugal. Samples were collected in summer and winter from the most contaminated sites from where the water flows downstream to the freshwater of Chança's river reservoir. The prokaryotic diversity on water bodies' sediments allowed us to distinguish the highly contaminated sites (pH ≈ 2) from sites with intermediate levels of contamination (pH ≈ 3-6.5), and from sites without contamination (pH ≈ 7.5). The abundances of acidophiles of genera Acidiphilium, Acidibacter, Acidobacterium and Acidocella in the sediments were correlated with the level of acid mine drainage contamination. The two first genera were among the 30 most abundant prokaryotes in all contaminated samples, including one (SS2w), where the contamination was very diluted, thereby emphasizing the impact that such type of pollution can have in the microbial communities of sediments. In addition, the high abundances of archaeal taxa from class Thermoplasmata and of bacteria from family RCP1-48 in the sediments from the most contaminated site corroborate their importance in such ecosystems and a putative role in the generation of acid mine drainage.

Antimicrobial Effects of Black Soldier Fly and Yellow Mealworm Fats and Their Impact on Gut Microbiota of Growing Rabbits.

Simple SummaryThe use of insect lipids as an alternative ingredient is an emergent topic in animal nutrition due to their antimicrobial activities. The present study evaluated the in vitro antimicrobial activities of two insect fats (black soldier fly (Hermetia illucens (HI) fat and yellow mealworm (Tenebrio molitor (TM) fat) and their effect as a total substitute for dietary soybean oil in cecal fermentation and gut microbiota of growing rabbits. The obtained results showed the potential of HI and TM fats as an antibacterial feed ingredient with a positive influence on the rabbit cecal microbiota. HI and TM fats therefore may be a sustainable lipid alternative to soybean oil in rabbit nutrition with possible interesting applications in the feed industry.This study aimed to evaluate the in vitro antimicrobial activities of two types of insect fats extracted from black soldier fly larvae (HI, Hermetia illucens L.) and yellow mealworm larvae (TM, Tenebrio molitor L.) and their effects as dietary replacement of soybean oil (S) on cecal fermentation pattern, and fecal and cecal microbiota in rabbits. A total of 120 weaned rabbits were randomly allotted to three dietary treatments (40 rabbits/group) —a control diet (C diet) containing 1.5% of S and two experimental diets (HI diet (HID) and TM diet (TMD)), where S was totally substituted by HI or TM fats during the whole trial that lasted 41 days. Regarding the in vitro antimicrobial activities, HI and TM fats did not show any effects on Salmonella growth. Yersinia enterocolitica showed significantly lower growth when challenged with HI fats than the controls. The insect fat supplementation in rabbit diets increased the contents of the cecal volatile fatty acids when compared to the control group. A metataxonomic approach was adopted to investigate the shift in the microbial composition as a function of the dietary insect fat supplementation. The microbiota did not show a clear separation as a function of the inclusion, even if a specific microbial signature was observed. Indeed, HI and TM fat supplementation enriched the presence of Akkermansia that was found to be correlated with NH3-N concentration. An increase in Ruminococcus, which can improve the immune response of the host, was also observed. This study confirms the potential of HI and TM fats as antibacterial feed ingredients with a positive influence on the rabbit cecal microbiota, thus supporting the possibility of including HI and TM fats in rabbit diets.

Portuguese cacholeira blood sausage: A first taste of its microbiota and volatile organic compounds

Among typical Portuguese sausages, the cacholeira blood sausage undoubtedly represents one of the most popular preparations. To the authors’ knowledge, a lack of information on both the microbiota and the volatile organic compounds (VOCs) of this blood-containing sausage emerges from the available scientific literature. This study represents the first characterization of physico-chemical, microbiological and volatile traits of Portuguese cacholeira blood sausage. To this end, ready-to-eat cacholeira blood sausages were collected from two production batches manufactured in summer (batch 1) and autumn (batch 2). Viable counts showed active microbial communities mainly composed by lactic acid bacteria, coagulase negative cocci, enterococci and eumycetes. The metataxonomic approach showed a simple bacterial composition, which was dominated by Lactobacillus sakei in both the analyzed batches (1 and 2) considered. Carnobacterium, Enterococcus, Kluyvera, Lactococcus and Serratia were found as minor genera. The mycobiota varied according to the production season. Batch 1 was dominated by Starmerella apicola, Debaryomyces hansenii and Candida tropicalis, whereas batch 2 was dominated by D. hansenii. Moreover, Aspergillus spp., Kurtzmaniella zeylanoides, Saccharomyces cerevisiae, Kurtzmaniella santamariae, Brettanomyces bruxellensis and Pichia kluyveri were detected in both the batches as minority species. Seventy-two volatile compounds were identified, including esters, phenols, terpenoids, acids, alcohols, ketones, aldehydes, lactones, furans, sulphur and nitrogen compounds. Significant differences were seen in the amount of some compounds, as a feasible consequence of differences in the raw materials, artisan production and seasonality.

Impact of Lactococcus lactis as starter culture on microbiota and metabolome profile of an Italian raw milk cheese

The standardisation of raw milk cheeses without losing their typicality is an ambitious goal. In the present study, the performance of two Lactococcus lactis autochthonous starter cultures (AS1 and AS2) was evaluated for the production of Valle d'Aosta (VdA, Italy) Fromadzo PDO, in terms of competition and persistence capacity (by rep-PCR strain characterisation), influence on indigenous microbial population (by metataxonomic approach) and volatilome development (by GC–MS and sensory analysis). The best performance was shown by AS2 starter mixture, which was able to colonise Fromadzo cheeses at the expense of indigenous lactic microbiota, and reached mean frequency values of 87% and 70% in 30 day-ripened products from valley floor and alpine pasture milk, respectively. Moreover, both kinds of cheeses, obtained using AS2, achieved the best scores in terms of aroma and taste, the pasture cheese also achieving the best score for texture.

Human Milk Microbiome and Maternal Postnatal Psychosocial Distress.

Human milk contains many bioactive components, including bacteria, which are transferred to the developing infant through breastfeeding. Milk bacteria appear to, amongst others, originate from the maternal gut. A mother’s postnatal psychosocial distress may alter maternal gut microbiota, which in turn may affect the bacteria present in milk. The aim of this study was to explore whether maternal postnatal psychosocial distress was related to alterations in the relative abundances of specific bacteria and to milk microbial diversity. Healthy mothers (N = 77; N = 51 with complete data) collected breast milk samples at 2, 6, and 12 weeks postpartum and filled in mood questionnaires on experienced stress, anxiety, and depressive symptoms at 6 weeks postpartum. A metataxonomic approach (16S rRNA gene sequencing (region V3 and V4) using Illumina MiSeq technology) was used to assess bacterial abundances and diversity. For the group as a whole, an increase in diversity of the milk bacterial community was observed during the first 3 months of breastfeeding (Shannon index). This general increase in diversity appears to be explained by an increase of Lactobacillus and other minor genera, together with a decrease in Staphylococcus. With respect to psychological distress and milk microbial composition, no significant differences in the relative abundance of major bacterial genera were detected between women with high (N = 13) and low (N = 13) psychosocial distress. However, progressive and distinct changes in the content of Firmicutes, Proteobacteria, and Bacteroidetes at the phylum level and Acinetobacter, Flavobacterium, and Lactobacillus at the genera level were observed in milk samples of women with low psychosocial distress. With respect to milk microbial diversity, high maternal psychosocial distress, compared to low maternal psychosocial distress, was related to significantly lower bacterial diversity in milk at 3 months post-delivery. Anxiety, stress, and depressive symptoms separately were unrelated to specific bacterial profiles. The current study suggests a potential relation between maternal psychosocial distress and milk microbiota, providing first evidence of a possible mechanism through which post-partum psychological symptoms may affect infant development and health.

Analysis of bacterial microbiome associated with nylon and copper nets in an aquaculture context

Bacterial pathogens represent a huge challenge for aquaculture profitability and fish welfare, particularly since they can naturally occur in the water column and fish microbiomes, appear associated with biofouling or establish biofilms on tanks, gear and nets. Recently, several studies have shown how biofilms can become important pathogen reservoirs within fish farms, particularly since they confer protection from external stressors, such as antibiotics, and can potentially favor virulence. Copper nets are presently regarded as a good alternative to other traditional netting material, such as nylon, due to their high durability and low maintenance, no fouling establishment and also due to the antimicrobial properties of copper. Here we compared the bacterial microbiomes associated with nylon and copper nets in an aquaculture context, using a metataxonomic approach through the high-throughput sequencing of the 16S rRNA V4 hypervariable region. Bacterial alpha-diversity associated with nylon was significantly higher than the associate with copper nets. Similarly, there was a significant effect of net type on bacterial beta-diversity. Additionally, among the most abundant bacteria colonizing copper nets there were taxa belonging to Tenacibaculum and Winogradskyella, which are both known to harbor potential fish pathogens. We thus highlight the importance of monitoring the composition of bacterial communities established in aquaculture gear, including copper nets, with future studies evaluating whether they may serve as reservoirs for pathogenic bacterial strains.

A Metataxonomic Tool to Investigate the Diversity of Treponema.

The genus Treponema contains a number of human and animal pathogenic as well as symbiotic bacteria that are found in vastly different anatomical and environmental habitats. Our understanding of the species range, evolution, and biology of these important bacteria is still limited. To explore the diversity of treponemes, we established, validated, and tested a novel metataxonomic approach. As the informative nature of the hypervariable regions of the 16S rRNA gene differ, we first analyzed each variable region independently. Considering the in silico results obtained, we established and validated the sequencing of the V4-region of the 16S rRNA gene using known mixtures of Treponema species as well as a selected number of clinical samples. The metataxonomic approach was able to identify Treponema to a near-species level. We demonstrate that using a spirochete-specific enrichment, our method is applicable to complex microbial communities and large variety of biological samples. The metataxonomic approach described provides a useful method to unravel the full diversity and range of Treponema in various ecosystems.

Meta-taxonomic analysis of prokaryotic and eukaryotic gut flora in stool samples from visceral leishmaniasis cases and endemic controls in Bihar State India.

Visceral leishmaniasis (VL) caused by Leishmania donovani remains of public health concern in rural India. Those at risk of VL are also at risk of other neglected tropical diseases (NTDs) including soil transmitted helminths. Intestinal helminths are potent regulators of host immune responses sometimes mediated through cross-talk with gut microbiota. We evaluate a meta-taxonomic approach to determine the composition of prokaryotic and eukaryotic gut microflora using amplicon-based sequencing of 16S ribosomal RNA (16S rRNA) and 18S rRNA gene regions. The most abundant bacterial taxa identified in faecal samples from Bihar State India were Prevotella (37.1%), Faecalibacterium (11.3%), Escherichia-Shigella (9.1%), Alloprevotella (4.5%), Bacteroides (4.1%), Ruminococcaceae UCG-002 (1.6%), and Bifidobacterium (1.5%). Eukaryotic taxa identified (excluding plant genera) included Blastocystis (57.9%; Order: Stramenopiles), Dientamoeba (12.1%; Family: Tritrichomonadea), Pentatrichomonas (10.1%; Family: Trichomonodea), Entamoeba (3.5%; Family: Entamoebida), Ascaridida (0.8%; Family: Chromodorea; concordant with Ascaris by microscopy), Rhabditida (0.8%; Family: Chromodorea; concordant with Strongyloides), and Cyclophyllidea (0.2%; Order: Eucestoda; concordant with Hymenolepis). Overall alpha (Shannon’s, Faith’s and Pielou’s indices) and beta (Bray-Curtis dissimilarity statistic; weighted UniFrac distances) diversity of taxa did not differ significantly by age, sex, geographic subdistrict, or VL case (N = 23) versus endemic control (EC; N = 23) status. However, taxon-specific associations occurred: (i) Ruminococcaceae UCG- 014 and Gastranaerophilales_uncultured bacterium were enriched in EC compared to VL cases; (ii) Pentatrichomonas was more abundant in VL cases than in EC, whereas the reverse occurred for Entamoeba. Across the cohort, high Escherichia-Shigella was associated with reduced bacterial diversity, while high Blastocystis was associated with high bacterial diversity and low Escherichia-Shigella. Individuals with high Blastocystis had low Bacteroidaceae and high Clostridiales vadin BB60 whereas the reverse held true for low Blastocystis. This scoping study provides useful baseline data upon which to develop a broader analysis of pathogenic enteric microflora and their influence on gut microbial health and NTDs generally.

Metataxonomic analyses reveal differences in aquifer bacterial community as a function of creosote contamination and its potential for contaminant remediation

Metataxonomic approach was used to describe the bacterial community from a creosote-contaminated aquifer and to access the potential for in situ bioremediation of the polycyclic aromatic hydrocarbons (PAHs) by biostimulation. In general, the wells with higher PAH contamination had lower richness and diversity than others, using the Shannon and Simpson indices. By the principal coordinate analysis (PCoA) it was possible to observe the clustering of the bacterial community of most wells in response of the presence of PAH contamination. The significance analysis using edgeR package of the R program showed variation in the abundance of some Operational Taxonomic Units (OTUs) of contaminated wells compared to uncontaminated ones. Taxons enriched in the contaminated wells were correlated positively (p < 0.05) with the hydrocarbons, according to redundancy analysis (RDA). All these enriched taxa have been characterized as PAH degrading agents, such as the genus Comamonas, Geobacter, Hydrocarboniphaga, Anaerolinea and Desulfomonile. Additionally, it was possible to predict, with the PICRUSt program, a greater proportion of pathways and genes related to the degradation of PAHs in the wells with higher contamination levels. We conclude that the contaminants promoted the enrichment of several groups of degrading bacteria in the area, which strengthens the feasibility of applying biostimulation as an aquifer remediation strategy.

Precise Fecal Microbiome of the Herbivorous Tibetan Antelope Inhabiting High-Altitude Alpine Plateau.

The metataxonomic approach combining 16S rRNA gene amplicon sequencing using the PacBio Technology with the application of the operational phylogenetic unit (OPU) approach, has been used to analyze the fecal microbial composition of the high-altitude and herbivorous Tibetan antelopes. The fecal samples of the antelope were collected in Hoh Xil National Nature Reserve, at an altitude over 4500 m, the largest depopulated zone in Qinghai-Tibetan Plateau, China, where non-native animals or humans may experience life-threatening acute mountain sickness. In total, 104 antelope fecal samples were enrolled in this study, and were clustered into 61,258 operational taxonomic units (OTUs) at an identity of 98.7% and affiliated with 757 OPUs, including 144 known species, 256 potentially new species, 103 potentially higher taxa within known lineages. In addition, 254 comprised sequences not affiliating with any known family, and the closest relatives were unclassified lineages of existing orders or classes. A total of 42 out of 757 OPUs conformed to the core fecal microbiome, of which four major lineages, namely, un-cultured Ruminococcaceae, Lachnospiraceae, Akkermansia, and Christensenellaceae were associated with human health or longevity. The current study reveals that the fecal core microbiome of antelope is mainly composited of uncultured bacteria. The most abundant core taxa, namely, uncultured Ruminococcaceae, uncultured Akkermansia, uncultured Bacteroides, uncultured Christensenellaceae, uncultured Mollicutes, and uncultured Lachnospiraceae, may represent new bacterial candidates at high taxa levels, and several may have beneficial roles in health promotion or anti-intestinal dysbiosis. These organisms should be further isolated and evaluated for potential effect on human health and longevity.

Tu1930 - A Prospective Multi-National Study of the Colorectal Cancer Mucosal Microbiome Reveals Specific Taxonomic Changes Indicative of Disease Stage and Prognosis

Introduction The colorectal cancer (CRC) microbiome is niche specific and individualised. Several putative driver organisms enriched on CRCs have been identified from human studies, but few data exist which properly account for important clinical variables in CRC. In this study, we used a meta-taxonomic approach to demonstrate how the CRC microbiome varies with disease stage, histological markers of prognosis and host molecular phenotypes. Method A prospective study was performed on patients undergoing colonoscopy and elective surgery for CRC at three hospitals in UK and Czech Republic. Tissue was sampled from tumours, adenomas, adjacent normal mucosa and mucosa from healthy colon controls. The V1-2 regions of the 16S rRNA gene were sequenced (Illumina MiSeq); data were processed in Mothur and analysed in Stamp and R. Species assignment was performed with NCBI BLAST for microbial genomes. False discovery rate p value correction accounted for multiple testing. Histological analysis and tumour molecular phenotyping were performed according to Royal College of Pathology guidelines. Results One hundred and ninety six patients were recruited: 158 CRC patients, 24 adenoma patients and 14 normal colon controls (median age 70; range 35–90). Tumours were staged as 6 T0, 4 T1, 23 T2, 97 T3, 27 T4; 99 N0, 40 N1, 27 N2; 6 M1. No significant differences were seen in diversity or taxonomy between the UK and Czech cohorts. Adenoma and healthy colon control samples were taxonomically indistinct. However, CRCs were characterised by reduced Shannon diversity (p Conclusion This large prospective analysis demonstrates that the CRC microbiome is stage-specific and appears to evolve with disease progression. We conclude that oral pathobionts which colonise advanced stage disease relate to markers of tumour prognosis, raising the possibility that they may be directly influencing tumour invasion. Disclosure of Interest None Declared

A Metataxonomic Approach Could Be Considered for Cattle Clinical Mastitis Diagnostics.

Mastitis is one of the most costly diseases affecting the dairy industry, and identification of the causative microorganism(s) is essential. Here, we report the use of next-generation sequencing of bacterial 16S rRNA genes for clinical mastitis diagnosis. We used 65 paired milk samples, collected from the mastitic and a contralateral healthy quarter of mastitic dairy cattle to evaluate the technique as a potential alternative to bacterial culture or targeted PCR. One large commercial dairy farm was used, with one trained veterinarian collecting the milk samples. The 16S rRNA genes were individually amplified and sequenced using the MiSeq platform. The MiSeq Reporter was used in order to analyze the obtained sequences. Cattle were categorized according to whether or not 1 of the 10 most abundant bacterial genera in the mastitic quarter exhibited an increase in relative abundance between the healthy and mastitic quarters equal to, or exceeding, twofold. We suggest that this increase in relative abundance is indicative of the genus being a causative mastitis pathogen. Well-known mastitis-causing pathogens such as Streptococcus uberis and Staphylococcus spp. were identified in most cattle. We were able to diagnose 53 out of the 65 studied cases and identify potential new mastitis pathogens such as Sneathia sanguinegens and Listeria innocua, which are difficult to identify by bacterial culture because of their fastidious nature.

Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification.

To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341–534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions.