Impact of root canal preparation using two single-file systems on the intra-radicular microbiome of teeth with primary apical periodontitis.

OBJECTIVES: This study aimed to describe the effects of two single-file systems on the diversity of the endodontic microbiome of teeth with primary asymptomatic apical periodontitis. MATERIALS AND METHODS: The root canals from single-rooted teeth with apical periodontitis were prepared using either the Reciproc Blue (RB) or the XP-endo Shaper (XPS) instrument system. The latter was followed by a supplementary step with the XP-endo Finisher (XPF) instrument. For irrigation, 5.25% sodium hypochlorite was used. Root canal samples were taken at the baseline (S1), after preparation (S2), and after the supplementary step (S3). DNA was extracted and subjected to high-throughput sequencing using the MiSeq Illumina platform. RESULTS: Samples from 10 teeth from the RB and 7 from the XPS group were subjected to DNA sequencing. Initial samples differed significantly from post-preparation samples in bacterial diversity, with no significant difference when comparing the two instrument systems. The most dominant phyla in S2 were Bacteroidetes, Proteobacteria, Firmicutes, Fusobacteria, and Actinobacteria. The same phyla were found to dominate baseline samples and samples taken after using XPF, but with differences in the ranking of the most dominant ones. At the genus level, the most dominant genera identified after RB instrumentation were Bacteroidaceae [G-1], Fusobacterium, and Staphylococcus, while the most dominant genera after XPS instrumentation were Fusobacterium and Porphyromonas. These genera were also dominant in the initial samples. CONCLUSIONS: Both treatment protocols had measurable effects on the root canal microbial diversity, with no significant differences between them. Most of the dominant taxa involved in the primary infection and probably in the aetiology of apical periodontitis were eliminated or substantially reduced. CLINICAL RELEVANCE: The most dominant taxa that persisted after instrumentation were Fusobacterium, Porphyromonas, Staphylococcus, and Bacteroidaceae [G-1].

Mycobiome structure does not affect field litter decomposition in Eucalyptus and Acacia plantations.

Mixed tree plantations have been studied because of their potential to improve biomass production, ecosystem diversity, and soil quality. One example is a mixture of Eucalyptus and Acacia trees, which is a promising strategy to improve microbial diversity and nutrient cycling in soil. We examined how a mixture of these species may influence the biochemical attributes and fungal community associated with leaf litter, and the effects on litter decomposition. We studied the litter from pure and mixed plantations, evaluating the effects of plant material and incubation site on the mycobiome and decomposition rate using litterbags incubated in situ. Our central hypothesis was litter fungal community would change according to incubation site, and it would interfere in litter decomposition rate. Both the plant material and the incubation locale significantly affected the litter decomposition. The origin of the litter was the main modulator of the mycobiome, with distinct communities from one plant species to another. The community changed with the incubation time but the incubation site did not influence the mycobiome community. Our data showed that litter and soil did not share the main elements of the community. Contrary to our hypothesis, the microbial community structure and diversity lacked any association with the decomposition rate. The differences in the decomposition pattern are explained basically as a function of the exchange of nitrogen compounds between the litter.

Multi-domain probiotic consortium as an alternative to chemical remediation of oil spills at coral reefs and adjacent sites.

BACKGROUND: Beginning in the last century, coral reefs have suffered the consequences of anthropogenic activities, including oil contamination. Chemical remediation methods, such as dispersants, can cause substantial harm to corals and reduce their resilience to stressors. To evaluate the impacts of oil contamination and find potential alternative solutions to chemical dispersants, we conducted a mesocosm experiment with the fire coral Millepora alcicornis, which is sensitive to environmental changes. We exposed M. alcicornis to a realistic oil-spill scenario in which we applied an innovative multi-domain bioremediator consortium (bacteria, filamentous fungi, and yeast) and a chemical dispersant (Corexit® 9500, one of the most widely used dispersants), to assess the effects on host health and host-associated microbial communities. RESULTS: The selected multi-domain microbial consortium helped to mitigate the impacts of the oil, substantially degrading the polycyclic aromatic and n-alkane fractions and maintaining the physiological integrity of the corals. Exposure to Corexit 9500 negatively impacted the host physiology and altered the coral-associated microbial community. After exposure, the abundances of certain bacterial genera such as Rugeria and Roseovarius increased, as previously reported in stressed or diseased corals. We also identified several bioindicators of Corexit 9500 in the microbiome. The impact of Corexit 9500 on the coral health and microbial community was far greater than oil alone, killing corals after only 4 days of exposure in the flow-through system. In the treatments with Corexit 9500, the action of the bioremediator consortium could not be observed directly because of the extreme toxicity of the dispersant to M. alcicornis and its associated microbiome. CONCLUSIONS: Our results emphasize the importance of investigating the host-associated microbiome in order to detect and mitigate the effects of oil contamination on corals and the potential role of microbial mitigation and bioindicators as conservation tools. Chemical dispersants were far more damaging to corals and their associated microbiome than oil, and should not be used close to coral reefs. This study can aid in decision-making to minimize the negative effects of oil and dispersants on coral reefs. Video abstract.

Estrogen induces shift in abundances of specific groups of the coral microbiome.

Synthetic estrogens such as ethinylestradiol (EE2) are persistent micropollutants that are not effectively removed from wastewater by conventional treatments. These contaminants are released into waterbodies, where they disrupt endocrine systems of organisms and cause harmful effects such as feminization, infertility, reproduction problems and genital malformations. The consequences of this pollution for key marine ecosystems such as coral reefs and their associated microbiomes are underexplored. We evaluated the effects of EE2 concentrations of 100 ng L-1 and 100 µg L-1 on the coral metaorganism Mussismilia harttii. The results indicated no effects on visible bleaching or Fv/Fm ratios in the corals during a 17-day microcosm experiment. However, next-generation sequencing of 16S rDNA revealed a statistically significant effect of high EE2 concentrations on OTU richness, and shifts in specific microbial groups after treatments with or without EE2. These groups might be bioindicators of early shifts in the metaorganism composition caused by EE2 contamination.

Short-term effect of Eucalyptus plantations on soil microbial communities and soil-atmosphere methane and nitrous oxide exchange.

Soil greenhouse gas (GHG) emissions are a significant environmental problem resulting from microbially-mediated nitrogen (N) and carbon (C) cycling. This study aimed to investigate the impact of Eucalyptus plantations on the structure and function of a soil microbial community, and how resulting alterations may be linked to GHG fluxes. We sampled and monitored two adjacent Eucalyptus plantations-a recently logged site that harbored new seedlings and an adult plantation-and compared them to a site hosting native vegetation. We used 16S rRNA gene sequencing and qPCR amplifications of key nitrogen and methane cycle genes to characterize microbial structure and functional gene abundance and compared our data with soil parameters and GHG fluxes. Both microbial community attributes were significantly affected by land use and logging of Eucalyptus plantations. The genes nosZ and archaeal amoA were significantly more abundant in native forest than in either young or old Eucalyptus plantations. Statistical analyses suggest that land use type has a greater impact on microbial community structure and functional gene abundance than Eucalyptus rotation. There was no correlation between GHG fluxes and shifts in microbial community, suggesting that microbial community structure and functional gene abundance are not the main drivers of GHG fluxes in this system.

Close Link Between Harmful Cyanobacterial Dominance and Associated Bacterioplankton in a Tropical Eutrophic Reservoir.

Cyanobacteria tend to become the dominant phytoplankton component in eutrophic freshwater environments during warmer seasons. However, general observations of cyanobacterial adaptive advantages in these circumstances are insufficient to explain the prevalence of one species over another in a bloom period, which may be related to particular strategies and interactions with other components of the plankton community. In this study, we present an integrative view of a mixed cyanobacterial bloom occurring during a warm, rainy period in a tropical hydropower reservoir. We used high-throughput sequencing to follow temporal shifts in the dominance of cyanobacterial genera and shifts in the associated heterotrophic bacteria community. The bloom occurred during late spring-summer and included two distinct periods. The first period corresponded to Microcystis aeruginosa complex (MAC) dominance with a contribution from Dolichospermum circinale; this pattern coincided with high water retention time and low transparency. The second period corresponded to Cylindrospermopsis raciborskii and Synechococcus spp. dominance, and the reservoir presented lower water retention time and higher water transparency. The major bacterial phyla were primarily Cyanobacteria and Proteobacteria, followed by Actinobacteria, Bacteroidetes, Verrucomicrobia, and Planctomycetes. Temporal shifts in the dominance of cyanobacterial genera were not only associated with physical features of the water but also with shifts in the associated heterotrophic bacteria. The MAC bloom was associated with a high abundance of Bacteroidetes, particularly Cytophagales. In the second bloom period, Planctomycetes increased in relative abundance, five Planctomycetes OTUs were positively correlated with Synechococcus or C. raciborskii OTUs. Our results suggest specific interactions of the main cyanobacterial genera with certain groups of the heterotrophic bacterial community. Thus, considering biotic interactions may lead to a better understanding of the shifts in cyanobacterial dominance.

Clone wars: asexual reproduction dominates in the invasive range of Tubastraea spp. (Anthozoa: Scleractinia) in the South-Atlantic Ocean.

Although the invasive azooxanthellate corals Tubastraea coccinea and T. tagusensis are spreading quickly and outcompeting native species in the Atlantic Ocean, there is little information regarding the genetic structure and path of introduction for these species. Here we present the first data on genetic diversity and clonal structure from these two species using a new set of microsatellite markers. High proportions of clones were observed, indicating that asexual reproduction has a major role in the local population dynamics and, therefore, represents one of the main reasons for the invasion success. Although no significant population structure was found, results suggest the occurrence of multiple invasions for T. coccinea and also that both species are being transported along the coast by vectors such as oil platforms and monobouys, spreading these invasive species. In addition to the description of novel microsatellite markers, this study sheds new light into the invasive process of Tubastraea.

Antarctic strict anaerobic microbiota from Deschampsia antarctica vascular plants rhizosphere reveals high ecology and biotechnology relevance.

The Antarctic soil microbial community has a crucial role in the growth and stabilization of higher organisms, such as vascular plants. Analysis of the soil microbiota composition in that extreme environmental condition is crucial to understand the ecological importance and biotechnological potential. We evaluated the efficiency of isolation and abundance of strict anaerobes in the vascular plant Deschampsia antarctica rhizosphere collected in the Antarctic's Admiralty Bay and associated biodiversity to metabolic perspective and enzymatic activity. Using anaerobic cultivation methods, we identified and isolated a range of microbial taxa whose abundance was associated with Plant Growth-Promoting Bacteria (PGPB) and presences were exclusively endemic to the Antarctic continent. Firmicutes was the most abundant phylum (73 %), with the genus Clostridium found as the most isolated taxa. Here, we describe two soil treatments (oxygen gradient and heat shock) and 27 physicochemical culture conditions were able to increase the diversity of anaerobic bacteria isolates. Heat shock treatment allowed to isolate a high percentage of new species (63.63 %), as well as isolation of species with high enzymatic activity (80.77 %), which would have potential industry application. Our findings contribute to the understanding of the role of anaerobic microbes regarding ecology, evolutionary, and biotechnological features essential to the Antarctic ecosystem.

Microbiome in the Apical Root Canal System of Teeth with Post-Treatment Apical Periodontitis.

INTRODUCTION: Bacteria present in the apical root canal system are directly involved with the pathogenesis of post-treatment apical periodontitis. This study used a next-generation sequencing approach to identify the bacterial taxa occurring in cryopulverized apical root samples from root canal-treated teeth with post-treatment disease. METHODS: Apical root specimens obtained during periradicular surgery of ten adequately treated teeth with persistent apical periodontitis were cryogenically ground. DNA was extracted from the powder and the microbiome was characterized on the basis of the V4 hypervariable region of the 16S rRNA gene by using paired-end sequencing on Illumina MiSeq device. RESULTS: All samples were positive for the presence of bacterial DNA. Bacterial taxa were mapped to 11 phyla and 103 genera composed by 538 distinct operational taxonomic units (OTUs) at 3% of dissimilarity. Over 85% of the sequences belonged to 4 phyla: Proteobacteria, Firmicutes, Fusobacteria and Actinobacteria. In general, these 4 phyla accounted for approximately 80% of the distinct OTUs found in the apical root samples. Proteobacteria was the most abundant phylum in 6/10 samples. Fourteen genera had representatives identified in all cases. Overall, the genera Fusobacterium and Pseudomonas were the most dominant. Enterococcus was found in 4 cases, always in relatively low abundance. CONCLUSIONS: This study showed a highly complex bacterial community in the apical root canal system of adequately treated teeth with persistent apical periodontitis. This suggests that this disease is characterized by multispecies bacterial communities and has a heterogeneous etiology, because the community composition largely varied from case to case.

Microbiome of Deep Dentinal Caries Lesions in Teeth with Symptomatic Irreversible Pulpitis.

This study used a next-generation sequencing approach to identify the bacterial taxa occurring in the advanced front of caries biofilms associated with pulp exposure and irreversible pulpitis. Samples were taken from the deepest layer of dentinal caries lesions associated with pulp exposure in 10 teeth diagnosed with symptomatic irreversible pulpitis. DNA was extracted and the microbiome was characterized on the basis of the V4 hypervariable region of the 16S rRNA gene by using paired-end sequencing on Illumina MiSeq device. Bacterial taxa were mapped to 14 phyla and 101 genera composed by 706 different OTUs. Three phyla accounted for approximately 98% of the sequences: Firmicutes, Actinobacteria and Proteobacteria. These phyla were also the ones with most representatives at the species level. Firmicutes was the most abundant phylum in 9/10 samples. As for genera, Lactobacillus accounted for 42.3% of the sequences, followed by Olsenella (13.7%), Pseudoramibacter (10.7%) and Streptococcus (5.5%). Half of the samples were heavily dominated by Lactobacillus, while in the other half lactobacilli were in very low abundance and the most dominant genera were Pseudoramibacter, Olsenella, Streptococcus, and Stenotrophomonas. High bacterial diversity occurred in deep dentinal caries lesions associated with symptomatic irreversible pulpitis. The microbiome could be classified according to the relative abundance of Lactobacillus. Except for Lactobacillus species, most of the highly prevalent and abundant bacterial taxa identified in this study have been commonly detected in infected root canals. The detected taxa can be regarded as candidate pathogens for irreversible pulpitis and possibly the pioneers in pulp invasion to initiate endodontic infection.

Intercropped silviculture systems, a key to achieving soil fungal community management in eucalyptus plantations.

Fungi are ubiquitous and important contributors to soil nutrient cycling, playing a vital role in C, N and P turnover, with many fungi having direct beneficial relationships with plants. However, the factors that modulate the soil fungal community are poorly understood. We studied the degree to which the composition of tree species affected the soil fungal community structure and diversity by pyrosequencing the 28S rRNA gene in soil DNA. We were also interested in whether intercropping (mixed plantation of two plant species) could be used to select fungal species. More than 50,000 high quality sequences were analyzed from three treatments: monoculture of Eucalyptus; monoculture of Acacia mangium; and a mixed plantation with both species sampled 2 and 3 years after planting. We found that the plant type had a major effect on the soil fungal community structure, with 75% of the sequences from the Eucalyptus soil belonging to Basidiomycota and 19% to Ascomycota, and the Acacia soil having a sequence distribution of 28% and 62%, respectively. The intercropping of Acacia mangium in a Eucalyptus plantation significantly increased the number of fungal genera and the diversity indices and introduced or increased the frequency of several genera that were not found in the monoculture cultivation samples. Our results suggest that management of soil fungi is possible by manipulating the composition of the plant community, and intercropped systems can be a means to achieve that.

Impact of next generation sequencing techniques in food microbiology.

Understanding the Maxam-Gilbert and Sanger sequencing as the first generation, in recent years there has been an explosion of newly-developed sequencing strategies, which are usually referred to as next generation sequencing (NGS) techniques. NGS techniques have high-throughputs and produce thousands or even millions of sequences at the same time. These sequences allow for the accurate identification of microbial taxa, including uncultivable organisms and those present in small numbers. In specific applications, NGS provides a complete inventory of all microbial operons and genes present or being expressed under different study conditions. NGS techniques are revolutionizing the field of microbial ecology and have recently been used to examine several food ecosystems. After a short introduction to the most common NGS systems and platforms, this review addresses how NGS techniques have been employed in the study of food microbiota and food fermentations, and discusses their limits and perspectives. The most important findings are reviewed, including those made in the study of the microbiota of milk, fermented dairy products, and plant-, meat- and fish-derived fermented foods. The knowledge that can be gained on microbial diversity, population structure and population dynamics via the use of these technologies could be vital in improving the monitoring and manipulation of foods and fermented food products. They should also improve their safety.

Microbial diversity of a Mediterranean soil and its changes after biotransformed dry olive residue amendment.

The Mediterranean basin has been identified as a biodiversity hotspot, about whose soil microbial diversity little is known. Intensive land use and aggressive management practices are degrading the soil, with a consequent loss of fertility. The use of organic amendments such as dry olive residue (DOR), a waste produced by a two-phase olive-oil extraction system, has been proposed as an effective way to improve soil properties. However, before its application to soil, DOR needs a pre-treatment, such as by a ligninolytic fungal transformation, e.g. Coriolopsis floccosa. The present study aimed to describe the bacterial and fungal diversity in a Mediterranean soil and to assess the impact of raw DOR (DOR) and C. floccosa-transformed DOR (CORDOR) on function and phylogeny of soil microbial communities after 0, 30 and 60 days. Pyrosequencing of the 16S rRNA gene demonstrated that bacterial diversity was dominated by the phyla Proteobacteria, Acidobacteria, and Actinobacteria, while 28S-rRNA gene data revealed that Ascomycota and Basidiomycota accounted for the majority of phyla in the fungal community. A Biolog EcoPlate experiment showed that DOR and CORDOR amendments decreased functional diversity and altered microbial functional structures. These changes in soil functionality occurred in parallel with those in phylogenetic bacterial and fungal community structures. Some bacterial and fungal groups increased while others decreased depending on the relative abundance of beneficial and toxic substances incorporated with each amendment. In general, DOR was observed to be more disruptive than CORDOR.

Diversity of thermophilic bacteria in raw, pasteurized and selectively-cultured milk, as assessed by culturing, PCR-DGGE and pyrosequencing.

Thermophilic lactic acid bacteria (LAB) species, such as Streptococcus thermophilus, Lactobacillus delbrueckii and Lactobacillus helveticus, enjoy worldwide economic importance as dairy starters. To assess the diversity of thermophilic bacteria in milk, milk samples were enriched in thermophilic organisms through a stepwise procedure which included pasteurization of milk at 63 °C for 30 min (PM samples) and pasteurization followed by incubation at 42 °C for 24 h (IPM samples). The microbial composition of these samples was analyzed by culture-dependent (at 42 °C) and culture-independent (PCR-DGGE and pyrosequencing of 16S rRNA gene amplicons) microbial techniques. The results were then compared to those obtained for their corresponding starting raw milk counterparts (RM samples). Twenty different species were scored by culturing among 352 isolates purified from the counting plates and identified by molecular methods. Mesophilic LAB species (Lactococcus lactis, Lactococcus garvieae) were dominant (87% of the isolates) among the RM samples. However, S. thermophilus and Lb. delbrueckii were found to be the dominant recoverable organisms in both PM and IPM samples. The DGGE profiles of RM and PM samples were found to be very similar; the most prominent bands belonging to Lactococcus, Leuconostoc and Streptococcus species. In contrast, just three DGGE bands were obtained for IPM samples, two of which were assigned to S. thermophilus. The pyrosequencing results scored 95 operational taxonomic units (OTUs) at 3% sequence divergence in an RM sample, while only 13 were encountered in two IPM samples. This technique identified Leuconostoc citreum as the dominant microorganism in the RM sample, while S. thermophilus constituted more than 98% of the reads in the IPM samples. The procedure followed in this study allowed to estimate the bacterial diversity in milk and afford a suitable strategy for the isolation of new thermophilic LAB strains, among which adequate starters might be selected.

Effect of sugarcane burning or green harvest methods on the Brazilian Cerrado soil bacterial community structure.

BACKGROUND: The Brazilian Cerrado is one of the most important biodiversity reservoirs in the world. The sugarcane cultivation is expanding in this biome and necessitates the study of how it may impact the soil properties of the Cerrado. There is a lack of information especially about the impacts of different sugarcane management on the native bacterial communities of Cerrado soil. Therefore, our objective was to evaluate and compare the soil bacterial community structure of the Cerrado vegetation with two sugarcane systems. METHODS: We evaluated samples under native vegetation and the impact of the two most commonly used management strategies for sugarcane cultivation (burnt cane and green cane) on this diversity using pyrosequencing and quantitative PCR of the rrs gene (16S rRNA). RESULTS AND CONCLUSIONS: Nineteen different phyla were identified, with Acidobacteria (≈35%), Proteobacteria (≈24%) and Actinobacteria (≈21%) being the most abundant. Many of the sequences were represented by few operational taxonomic units (OTUs, 3% of dissimilarity), which were found in all treatments. In contrast, there were very strong patterns of local selection, with many OTUs occurring only in one sample. Our results reveal a complex bacterial diversity, with a large fraction of microorganisms not yet described, reinforcing the importance of this biome. As possible sign of threat, the qPCR detected a reduction of the bacterial population in agricultural soils compared with native Cerrado soil communities. We conclude that sugarcane cultivation promoted significant structural changes in the soil bacterial community, with Firmicutes phylum and Acidobacteria classes being the groups most affected.

Physical-chemical and microbiological changes in Cerrado Soil under differing sugarcane harvest management systems.

BACKGROUND: Sugarcane cultivation plays an important role in Brazilian economy, and it is expanding fast, mainly due to the increasing demand for ethanol production. In order to understand the impact of sugarcane cultivation and management, we studied sugarcane under different management regimes (pre-harvest burn and mechanical, unburnt harvest, or green cane), next to a control treatment with native vegetation. The soil bacterial community structure (including an evaluation of the diversity of the ammonia oxidizing (amoA) and denitrifying (nirK) genes), greenhouse gas flow and several soil physicochemical properties were evaluated. RESULTS: Our results indicate that sugarcane cultivation in this region resulted in changes in several soil properties. Moreover, such changes are reflected in the soil microbiota. No significant influence of soil management on greenhouse gas fluxes was found. However, we did find a relationship between the biological changes and the dynamics of soil nutrients. In particular, the burnt cane and green cane treatments had distinct modifications. There were significant differences in the structure of the total bacterial, the ammonia oxidizing and the denitrifying bacterial communities, being that these groups responded differently to the changes in the soil. A combination of physical and chemical factors was correlated to the changes in the structures of the total bacterial communities of the soil. The changes in the structures of the functional groups follow a different pattern than the physicochemical variables. The latter might indicate a strong influence of interactions among different bacterial groups in the N cycle, emphasizing the importance of biological factors in the structuring of these communities. CONCLUSION: Sugarcane land use significantly impacted the structure of total selected soil bacterial communities and ammonia oxidizing and denitrifier gene diversities in a Cerrado field site in Central Brazil. A high impact of land use was observed in soil under the common burnt cane management. The green cane soil also presented different profiles compared to the control soil, but to at a lesser degree.