Regulatory Effect of Irresistin-16 on Competitive Dual-Species Biofilms Composed of Streptococcus mutans and Streptococcus sanguinis

Based on the ecological plaque hypothesis, suppressing opportunistic pathogens within biofilms, rather than killing microbes indiscriminately, could be a biofilm control strategy for managing dental caries. The present study aimed to evaluate the effects of irresistin-16 (IRS-16) on competitive dual-species biofilms, which consisted of the conditional cariogenic agent Streptococcus mutans (S. mutans) and oral commensal bacteria Streptococcus sanguinis (S. sanguinis). Bacterial growth and biofilm formation were monitored using growth curve and crystal violet staining, respectively. The microbial proportion was determined using fluorescence in situ hybridization. A 2, 5-diphenyltetrazolium bromide assay was used to measure the metabolic activity of biofilms. Bacterial/extracellular polysaccharide (EPS) dyeing, together with water-insoluble EPS measurements, were used to estimate EPS synthesis. A lactic acid assay was performed to detect lactic acid generation in biofilms. The cytotoxicity of IRS-16 was evaluated in mouse fibroblast L929 cells using a live/dead cell viability assay and cell counting kit-8 assay. Our results showed that IRS-16 exhibited selective anti-biofilm activity, leading to a remarkable survival disadvantage of S. mutans within competitive dual-species biofilms. In addition, the metabolic activity, EPS synthesis, and acid generation of dual-species biofilms were significantly reduced by IRS-16. Moreover, IRS-16 showed minimal cytotoxicity against mouse fibroblast L929 cells. In conclusion, IRS-16 exhibited remarkable regulatory effects on dual-species biofilms composed of S. mutans and S. sanguinis with low cytotoxicity, suggesting that it may have potential for use in caries management through ecological biofilm control.

Soil Acidification, Mineral Neoformation and Heavy Metal Contamination Driven by Weathering of Sulphide Wastes in a Ramsar Wetland

Past waste disposal practices have left large volumes of sulphidic material stockpiled in a Ramsar wetland site on the Atlantic coast of southwestern Spain, leading to severe land degradation. With the aim of addressing this legacy issue, soil core samples were collected along two transects extending from the abandoned stockpiles to the adjacent marshland and subjected to XRD, SEM-EDS, ICP-OES and ICP-MS analyses. Sulphide oxidation has been shown to be a major driver of acid generation and metal leaching into the environment. The marsh soil receiving acid discharges from the sulphide wastes contains elevated levels (in mg kg−1) of Pb (up to 9838), As (up to 1538), Zn (up to 1486), Cu (up to 705), Sb (up to 225) and Tl (up to 13), which are retained both in relatively insoluble secondary minerals (mainly metal sulphates and oxides) and in easily soluble hydrated salts that serve as a transitory pool of acidity and available metals. By using a number of enrichment calculation methods that relate the metal concentrations in soil and their baseline concentrations and regulatory thresholds, there is enough evidence to conclude that these pollutants may pose an unacceptable risk to human and ecological receptors.

Small-scale field evaluation of geochemical blending of waste rock to mitigate acid rock drainage potential

Blending of potentially acid generating (PAG) waste rock with non-PAG waste rock to create a rock mixture which performs as non-PAG is a possible approach to permanent prevention of acid rock drainage (ARD) for PAG waste rock. In 2012, a field weathering study using 300 kg samples was implemented at Teck Coal's Quintette Project located in northeastern British Columbia, Canada to test the prevention of acid generation in the PAG waste rock by dissolved carbonate leached from overlying non-PAG waste rock and direct neutralization of acidic water from PAG waste rock by contact with non-PAG waste rock.After eight years of monitoring the experiments, the layered non-PAG on PAG barrels provided proof-of-concept that as the thickness of the PAG layer increases relative to the thickness of the non-PAG layers, acidic waters are more likely to be produced. The PAG on non-PAG layering has resulted in non-acidic water and no indications of metal leaching despite accelerated oxidation in the PAG layer shown by sulphate loadings. The study has demonstrated that the scale of heterogeneity of PAG and non-PAG materials is a critical consideration for providing certainty that rock blends designed to be non-PAG will perform as non-PAG in perpetuity. This is contrary to the standard paradigm in which an excess of acid-consuming minerals is often considered sufficient alone to ensure ARD is not produced.

Genomic insights into the ecological role and evolution of a novel Thermoplasmata order, “ Candidatus Sysuiplasmatales”

Recent omics studies have provided invaluable insights into the metabolic potential, adaptation and evolution of novel archaeal lineages from a variety of extreme environments. We have utilized a genome-resolved metagenomic approach to recover eight medium- to high-quality metagenome-assembled genomes (MAGs) that likely represent a new order (“ Candidatus Sysuiplasmatales”) within Thermoplasmata from mine tailings and acid mine drainage (AMD) sediments sampled from two copper mines in South China. 16S rRNA gene based analyses revealed a narrow habitat range for these uncultured archaea limiting to AMD and hot spring-related environments. Metabolic reconstruction indicated a facultatively anaerobic heterotrophic lifestyle. This may allow the archaea to adapt to oxygen fluctuations and is thus in marked contrast to the majority of lineages in the domain Archaea which typically show obligately anaerobic metabolisms. Notably, “ Ca. Sysuiplasmatales” could conserve energy through degradation of fatty acids, amino acid metabolism and oxidation of reduced inorganic sulfur compounds (RISCs), suggesting that they may contribute to acid generation in the extreme mine environments. Unlike its closely related Methanomassiliicoccales and “ Ca. Gimiplasmatales”, “ Ca. Sysuiplasmatales” lack the capacity to perform methanogenesis and carbon fixation. Ancestral state reconstruction indicated that “ Ca. Sysuiplasmatales” and its closely related Methanomassiliicoccales, “ Ca. Gimiplasmatales”, and the SG8-5 and the RBG-16-68-12 orders originated from a facultatively anaerobic ancestor capable of carbon fixation via the bacterial-type H 4 F Wood–Ljungdahl pathway (WLP). Their metabolic divergence might be attributed to different evolutionary paths. Importance A wide array of archaea populate Earth’s extreme environments thereby they may play important roles in mediating biogeochemical processes such as iron and sulfur cycling. However, our knowledge of archaeal biology and evolution is still limited considering the uncultured majority of archaeal diversity. For instance, most order-level lineages except Thermoplasmatales, Aciduliprofundales and Methanomassiliicoccales within Thermoplasmata do not have cultured representatives. Here, we report the discovery and genomic characterization of a novel order, namely “ Ca . Sysuiplasmatales”, within Thermoplasmata in the extremely acidic mine environments. “ Ca . Sysuiplasmatales” are inferred to be facultatively anaerobic heterotrophs and likely contribute to acid generation through the oxidation of RISCs. The physiological divergence between “ Ca . Sysuiplasmatales” and its closely related Thermoplasmata lineages may be attributed to different evolutionary paths. These results expand our knowledge of archaea in the extreme mine ecosystem.