Assessing the strength and sensitivity of the core microbiota approach on a highly diverse sponge reef.

Marine sponge reefs usually comprise a complex array of taxonomically different sponge species, many of these hosting highly diverse microbial communities. The number of microbial species known to occupy a given sponge ranges from tens to thousands, bringing numerous challenges to their analysis. One way to deal with such complexity is to use a core microbiota approach, in which only prevalent and abundant microbes are considered. Here we aimed to test the strength and sensitivity of the core microbiota approach by applying different core definitions to 20 host sponge species. Application of increasingly stringent relative abundance and/or percentage occurrence thresholds to qualify as part of the core microbiota decreased the number of 'core' OTUs and phyla and, consequently, changed both alpha- and beta-diversity patterns. Moreover, microbial co-occurrence patterns explored using correlation networks were also affected by the core microbiota definition. The application of stricter thresholds resulted in smaller and less compartmentalized networks, with different keystone species. These results highlight that the application of different core definitions to phylogenetically disparate host species can result in the drawing of markedly different conclusions. Consequently, we recommend to assess the effects of different core community definitions on the specific system of study before considering its application.

A non-invasive method to monitor marine pollution from bacterial DNA present in fish skin mucus.

Marine coastal contamination caused by human activity is a major issue worldwide. The implementation of effective pollution monitoring programs, especially in coastal areas, is important and urgent. The use of biological, physiological, or biochemical measurements to monitor the impacts of pollution has garnered increasing interest, particularly for the development of new non-invasive tools to assess water pollution. Fish skin mucus is in direct contact with the marine environment, making it a favourable microenvironment for the formation of biofilm bacterial communities. In this study, we developed a non-invasive technique, sampling fish skin mucus to determine and analyse bacterial community composition using next-generation sequencing. We hypothesised that bacterial communities associated with the skin mucus of a common harbour benthic blennioid triplefin fish, Forsterygion capito, would reflect conditions of different marine environments. We detected clear differences in bacterial community alpha-diversity between contaminated and reference sites. Beta-diversity analysis also revealed differences in the bacterial community structure of the skin mucus of fish inhabiting different geographical areas. The relative abundance of different bacterial orders varied among sites, as determined by linear discriminant analysis (LDA) and effect size (LEfSe) analyses. The observed variation in bacterial community compositions correlated more strongly with variation in hydrocarbons than to various metal concentrations. Using advanced DNA sequencing technologies, we have developed a novel non-invasive, low-cost and effective tool to monitor the impacts of pollution through analysis of the bacterial communities associated with fish skin mucus.

Using carbon substrate as a selection pressure to enhance the potential of aerobic granular sludge microbial communities for removing contaminants of emerging concern.

The ability of aerobic granular sludge (AGS) technology to biotransform contaminants of emerging concern (CECs) is largely unknown. AGS supplemented with either acetate, 2-propanol, glycerol, or a 1:1:1 mixture of all three, were cultivated to investigate the link between carbon supplements and biotransformation of six CECs. Carbon substrate had a significant effect on the microbial community composition, as assessed by 16S rRNA gene sequence analyses. Substrate degradation requiring a larger number of catabolic reactions (i.e., glycerol and the mix) was associated with greater microbial richness. The biotransformation of CECs was 45.9% greater in communities supplemented with glycerol (60.3 ±â€¯30.2 µg L-1 VSS-1) compared to acetate (20.9 ±â€¯29.7 µg L-1 VSS-1). Database surveys of metabolic reactions indicate that microbial communities supplemented with glycerol have the greatest capacity for the degradation of aromatic compounds, while those supplemented with acetate community have the lowest.

Vertical transmission of sponge microbiota is inconsistent and unfaithful.

Co-evolutionary theory predicts that if beneficial microbial symbionts improve host fitness, they should be faithfully transmitted to offspring. More recently, the hologenome theory of evolution predicts resemblance between parent and offspring microbiomes and high partner fidelity between host species and their vertically transmitted microbes. Here, we test these ideas in multiple coexisting host species with highly diverse microbiota, leveraging known parent-offspring pairs sampled from eight species of wild marine sponges (Porifera). We found that the processes governing vertical transmission were both neutral and selective. A neutral model was a better fit to larval (R2 = 0.66) than to the adult microbiota (R2 = 0.27), suggesting that the importance of non-neutral processes increases as the sponge host matures. Microbes that are enriched above neutral expectations in adults were disproportionately transferred to offspring. Patterns of vertical transmission were, however, incomplete: larval sponges shared, on average, 44.8% of microbes with their parents, which was not higher than the fraction they shared with nearby non-parental adults. Vertical transmission was also inconsistent across siblings, as larval sponges from the same parent shared only 17% of microbes. Finally, we found no evidence that vertically transmitted microbes are faithful to a single sponge host species. Surprisingly, larvae were as likely to share vertically transmitted microbes with larvae from other sponge species as they were with their own species. Our study demonstrates that common predictions of vertical transmission that stem from species-poor systems are not necessarily true when scaling up to diverse and complex microbiomes.

Microbial assemblages and bioindicators as proxies for ecosystem health status: potential and limitations.

Microorganisms play fundamental roles in the diversity and functional stability of environments, including nutrient and energy cycling. However, microbial biodiversity loss and change because of global climate and land use change remain poorly understood. Many microbial taxa exhibit fast growth rates and are highly sensitive to environmental change. This suggests they have potential to be efficient biological indicators to assess and monitor the state of the habitats within which they occur. Here, we describe and illustrate a range of univariate and multivariate statistical approaches that can be used to identify effective microbial indicators of environmental perturbations and quantify changes in microbial communities. We show that the integration of multiple approaches, such as linear discriminant analysis effect size and indicator value analysis, is optimal for the quantification of the effects of perturbation on microbial communities. We demonstrate the most prevalent techniques using microbial community data derived from soils under different land uses. We discuss the limitations to the development and use of microbial bioindicators and identify future research directions, such as the creation of reliable, standardised reference databases to provide baseline metrics that are indicative of healthy microbial communities. If reliable and globally-relevant microbial indicators of environmental health can be developed, there is enormous potential for their use, both as a standalone monitoring tool and via their integration with existing physical, chemical and biological measures of environmental health.

Pathogen reservoir hypothesis investigated by analyses of the adenotonsillar and middle ear microbiota.

INTRODUCTION: Adenotonsillar and middle ear diseases result in some of the most frequently performed operations in the pediatric population worldwide. The pathogen reservoir hypothesis (PRH) suggests that the adenoids act as a reservoir of bacteria which play a potential pathogenic role in otitis media. Evidence supporting this hypothesis is limited. This study sought to comprehensively determine and compare associations between the adenotonsillar and middle ear bacterial microbiota within individual patients via next-generation sequencing and microbial network analyses. METHODS: Bacterial 16S rRNA gene-targeted amplicon sequencing was used to determine the bacterial composition of ten pediatric patients undergoing adenotonsillectomy and ventilation tube insertion for otitis media with effusion. At the time of surgery, swabs were taken from the adenoid surface, tonsil crypts and middle ear clefts (through the myringotomy incision). RESULTS: The most abundant sequences within the bacterial community at genus level across all anatomical sites were Fusobacterium, Haemophilus, Neisseria, and Porphyromonas. There was an observable difference in the relative abundance of bacterial communities, with a higher proportion of Haemophilus and Moraxella in the adenoid when compared with the middle ear. Furthermore, only one module (consisting of 4 bacterial OTUs) from one patient was identified through microbial network analyses to be significantly associated between middle ear and adenoid. In addition, microbial network analysis revealed that the adenoid and tonsil microbiota share greater similarity than do the adenoid and middle ear. CONCLUSION: The results of this study suggest that the adenoid microenvironment does not correlate to the middle ear microenvironment. A future study at the species level, and over time, is required to further investigate whether the differing relationship between the microbiota of the adenoid and middle ear rejects the pathogen reservoir hypothesis.

The bacterial community and local lymphocyte response are markedly different in patients with recurrent tonsillitis compared to obstructive sleep apnoea.

INTRODUCTION: Obstructive sleep apnea (OSA) is now a more common indication for tonsillectomy than recurrent tonsillitis (RT) [1,2]. Few studies have addressed possible differences in pathogenesis between these two conditions. Children with RT and OSA are often being treated in the community with multiple courses of antibiotics before surgery. Current understanding of the role of bacteria in disorders of the tonsils is mainly based on the culture of tonsil swabs. Swab cultures reflect only a very small fraction of the bacteria present on the mucosal surface and may not represent the bacterial communities within the tonsil crypts [3,4]. This study aimed to evaluate the local lymphocyte response and associations with bacterial community composition using molecular techniques of the tonsils removed from children for RT or OSA. METHOD: The palatine tonsils were removed by extracapsular dissection from 24 patients with age range one to ten years, 14 of whom had RT and 10 had OSA. The fixed tonsil tissues were evaluated for bacteria by Gram-staining and presence of connective tissue by safranin staining. B lymphocytes and T lymphocytes were also measured immunohistochemically. Finally, previously published bacterial community data for this cohort were reassessed for associations with RT and OSA, and with the observed lymphocyte patterns. RESULTS: In tonsils from patients with RT, large micro-colonies of bacteria were observed in the tonsil crypts, and a large number of B and T lymphocytes were noted immediately adjacent to the tonsil crypt itself. In marked contrast, the tonsils from patients with OSA had no bacteria identified, and no significant skewing of lymphocytes based on site (such as follicles or crypts). We observed that the majority of lymphocytes surrounding the bacterial micro-colonies were B lymphocytes with a mean ratio of 109:55 (B lymphocytes: T lymphocytes). Bacterial community diversity was not different between the two cohorts; however, there were significant differences in bacterial community composition. Children with RT had a higher relative abundance of members from the genera Parvimonas, Prevotella, and Treponema. While children with OSA had a higher relative abundance of Haemophilus, and Capnocytophaga. CONCLUSION: These results demonstrate significant differences in the local lymphocyte response and bacterial community composition in tonsil tissue between RT and OSA patients. It suggests that the response to antibiotics used in the treatment of these two conditions may be different. Furthermore, the presence of lymphocytes in RT within the tonsil crypt outside the tonsil epithelium is a unique observation of the location of these cells.

Paired analysis of the microbiota between surface tissue swabs and biopsies from pediatric patients undergoing adenotonsillectomy.

INTRODUCTION: Culture-independent methods, based on bacterial 16 S rRNA gene sequencing, have been used previously to investigate the adenotonsillar microbiota. However, these studies have focused on a single sampling site (usually a surface swab). We aimed to investigate potential differences in adenotonsillar microbiota according to sampling location, both on and within the adenoids and palatine tonsils. METHODS: Pediatric patients (n = 28, mean age five years) undergoing adenotonsillectomy were recruited for this study. At the time of surgery, a mucosal adenoid surface swab and an adenoid tissue biopsy was collected. Immediately following surgery, the crypts of the right and left tonsils were swabbed, and a surface and core tissue sample from the right tonsil were also collected. Bacterial 16 S rRNA gene-targeted amplicon sequencing was used to determine the bacterial composition of the collected samples. RESULTS: There was no significant difference in diversity or composition of the adenoid microbiota based on sampling site. However, the Shannon-Wiener and Inverse-Simpson diversity indices differed significantly (p < 0.05) between the microbial communities of the three different tonsil sampling sites. There was a higher average relative abundance of members from the genera Streptococcus, Actinobacillus, and Neisseria in the tonsil crypts when compared with surface and core tonsil tissue samples. CONCLUSION: Our results indicate that there is variation in bacterial diversity and composition based on sampling sites in the tonsils but not the adenoids. The difference in microbiota between the surface and the tissue may have implications for our understanding of the pathogenesis of recurrent tonsillitis and have treatment implications.

Phylogeny and genomics of SAUL, an enigmatic bacterial lineage frequently associated with marine sponges.

Many marine sponges contain dense and diverse communities of associated microorganisms. Members of the 'sponge-associated unclassified lineage' (SAUL) are frequently recorded from sponges, yet little is known about these bacteria. Here we investigated the distribution and phylogenetic status of SAUL. A meta-analysis of the available literature revealed the widespread distribution of this clade and its association with taxonomically varied sponge hosts. Phylogenetic analyses, conducted using both 16S rRNA gene-based phylogeny and concatenated marker protein sequences, revealed that SAUL is a sister clade of the candidate phylum 'Latescibacteria'. Furthermore, we conducted a comprehensive analysis of two draft genomes assembled from sponge metagenomes, revealing novel insights into the physiology of this symbiont. Metabolic reconstruction suggested that SAUL members are aerobic bacteria with facultative anaerobic metabolism, with the capacity to degrade multiple sponge- and algae-derived carbohydrates. We described for the first time in a sponge symbiont the putative genomic capacity to transport phosphate into the cell and to produce and store polyphosphate granules, presumably constituting a phosphate reservoir for the sponge host in deprivation periods. Our findings suggest that the lifestyle of SAUL is symbiotic with the host sponge, and identify symbiont factors which may facilitate the establishment and maintenance of this relationship.

The sponge microbiome project.

Marine sponges (phylum Porifera) are a diverse, phylogenetically deep-branching clade known for forming intimate partnerships with complex communities of microorganisms. To date, 16S rRNA gene sequencing studies have largely utilised different extraction and amplification methodologies to target the microbial communities of a limited number of sponge species, severely limiting comparative analyses of sponge microbial diversity and structure. Here, we provide an extensive and standardised dataset that will facilitate sponge microbiome comparisons across large spatial, temporal, and environmental scales. Samples from marine sponges (n = 3569 specimens), seawater (n = 370), marine sediments (n = 65) and other environments (n = 29) were collected from different locations across the globe. This dataset incorporates at least 268 different sponge species, including several yet unidentified taxa. The V4 region of the 16S rRNA gene was amplified and sequenced from extracted DNA using standardised procedures. Raw sequences (total of 1.1 billion sequences) were processed and clustered with (i) a standard protocol using QIIME closed-reference picking resulting in 39 543 operational taxonomic units (OTU) at 97% sequence identity, (ii) a de novo clustering using Mothur resulting in 518 246 OTUs, and (iii) a new high-resolution Deblur protocol resulting in 83 908 unique bacterial sequences. Abundance tables, representative sequences, taxonomic classifications, and metadata are provided. This dataset represents a comprehensive resource of sponge-associated microbial communities based on 16S rRNA gene sequences that can be used to address overarching hypotheses regarding host-associated prokaryotes, including host specificity, convergent evolution, environmental drivers of microbiome structure, and the sponge-associated rare biosphere.

Redefining the sponge-symbiont acquisition paradigm: sponge microbes exhibit chemotaxis towards host-derived compounds.

Marine sponges host stable and species-specific microbial symbionts that are thought to be acquired and maintained by the host through a combination of vertical transmission and filtration from the surrounding seawater. To assess whether the microbial symbionts also actively contribute to the establishment of these symbioses, we performed in situ experiments on Orpheus Island, Great Barrier Reef, to quantify the chemotactic responses of natural populations of seawater microorganisms towards cellular extracts of the reef sponge Rhopaloeides odorabile. Flow cytometry analysis revealed significant levels of microbial chemotaxis towards R. odorabile extracts and 16S rRNA gene amplicon sequencing showed enrichment of 'sponge-specific' microbial phylotypes, including a cluster within the Gemmatimonadetes and another within the Actinobacteria. These findings infer a potential mechanism for how sponges can acquire bacterial symbionts from the surrounding environment and suggest an active role of the symbionts in finding their host.

Evaluating the core microbiota in complex communities: A systematic investigation.

The study of complex microbial communities poses unique conceptual and analytical challenges, with microbial species potentially numbering in the thousands. With transient or allochthonous microorganisms often adding to this complexity, a 'core' microbiota approach, focusing only on the stable and permanent members of the community, is becoming increasingly popular. Given the various ways of defining a core microbiota, it is prudent to examine whether the definition of the core impacts upon the results obtained. Here we used complex marine sponge microbiotas and undertook a systematic evaluation of the degree to which different factors used to define the core influenced the conclusions. Significant differences in alpha- and beta-diversity were detected using some but not all core definitions. However, findings related to host specificity and environmental quality were largely insensitive to major changes in the core microbiota definition. Furthermore, none of the applied definitions altered our perception of the ecological networks summarising interactions among bacteria within the sponges. These results suggest that, while care should still be taken in interpretation, the core microbiota approach is surprisingly robust, at least for comparing microbiotas of closely related samples.

Diversity, structure and convergent evolution of the global sponge microbiome.

Sponges (phylum Porifera) are early-diverging metazoa renowned for establishing complex microbial symbioses. Here we present a global Porifera microbiome survey, set out to establish the ecological and evolutionary drivers of these host-microbe interactions. We show that sponges are a reservoir of exceptional microbial diversity and major contributors to the total microbial diversity of the world's oceans. Little commonality in species composition or structure is evident across the phylum, although symbiont communities are characterized by specialists and generalists rather than opportunists. Core sponge microbiomes are stable and characterized by generalist symbionts exhibiting amensal and/or commensal interactions. Symbionts that are phylogenetically unique to sponges do not disproportionally contribute to the core microbiome, and host phylogeny impacts complexity rather than composition of the symbiont community. Our findings support a model of independent assembly and evolution in symbiont communities across the entire host phylum, with convergent forces resulting in analogous community organization and interactions.