Diversity of fungi associated with petroglyph sites in the Negev Desert, Israel, and their potential role in bioweathering.

The petroglyphs of the Negev Desert, Israel, are famous and valuable archaeological remains. Previous studies have investigated the microbial communities associated with petroglyphs and their potential role in stone deterioration; nevertheless, the role of fungi remains unclear. In this study, the fungal communities present on the stone and, as a comparison, in the surrounding environment (soil and air) at Negev petroglyph sites were analyzed by means of culture-dependent and -independent (metagenomic) techniques. The metagenomic results showed a high fungal biodiversity in the soil, and both approaches highlighted the prevalence of species producing melanized, large, thick-walled spores (mainly Alternaria spp.). From the air sampling, mostly Cladosporium spp. were retrieved. On the other hand, on the rock, the results seem to indicate a low presence of fungi, but with a rock-specialized mycobiota consisting of extremotolerant microcolonial fungi (MCF) (e.g., Vermiconidia and Coniosporium) and lichens (Flavoplaca). In addition, low proportions of cosmopolitan fungi were detected on the stone, but the comparison of the data clearly indicates that they are transients from the surrounding environment. The ability of the isolated strains to dissolve CaCO3 and therefore be a potential threat to the petroglyphs (limestone substrate) was tested, but only one strain resulted in positive acid production under laboratory conditions. Nevertheless, both lichens and MCF detected in this study are well-known stone deteriogens, which may have a significant impact on the petroglyph's deterioration.

Microbial dark matter sequences verification in amplicon sequencing and environmental metagenomics data.

Although microorganisms constitute the most diverse and abundant life form on Earth, in many environments, the vast majority of them remain uncultured. As it is based on information gleaned mainly from cultivated microorganisms, our current body of knowledge regarding microbial life is partial and does not reflect actual microbial diversity. That diversity is hidden in the uncultured microbial majority, termed by microbiologists as "microbial dark matter" (MDM), a term borrowed from astrophysics. Metagenomic sequencing analysis techniques (both 16S rRNA gene and shotgun sequencing) compare gene sequences to reference databases, each of which represents only a small fraction of the existing microorganisms. Unaligned sequences lead to groups of "unknown microorganisms" that are usually ignored and rarefied from diversity analysis. To address this knowledge gap, we analyzed the 16S rRNA gene sequences of microbial communities from four different environments-a living organism, a desert environment, a natural aquatic environment, and a membrane bioreactor for wastewater treatment. From those datasets, we chose representative sequences of potentially unknown bacteria for additional examination as "microbial dark matter sequences" (MDMS). Sequence existence was validated by specific amplification and re-sequencing. These sequences were screened against databases and aligned to the Genome Taxonomy Database to build a comprehensive phylogenetic tree for additional sequence classification, revealing potentially new candidate phyla and other lineages. These putative MDMS were also screened against metagenome-assembled genomes from the explored environments for additional validation and for taxonomic and metabolic characterizations. This study shows the immense importance of MDMS in environmental metataxonomic analyses of 16S rRNA gene sequences and provides a simple and readily available methodology for the examination of MDM hidden behind amplicon sequencing results.

The Rise and Fall of Omicron BA.1 Variant as Seen in Wastewater Supports Epidemiological Model Predictions.

The COVID-19 pandemic caused by the SARS-CoV-2 virus has inflicted significant mortality and morbidity worldwide. Continuous virus mutations have led to the emergence of new variants. The Omicron BA.1 sub-lineage prevailed as the dominant variant globally at the beginning of 2022 but was subsequently replaced by BA.2 in numerous countries. Wastewater-based epidemiology (WBE) offers an efficient tool for capturing viral shedding from infected individuals, enabling early detection of potential pandemic outbreaks without relying solely on community cooperation and clinical testing resources. This study integrated RT-qPCR assays for detecting general SARS-CoV-2 and its variants levels in wastewater into a modified triple susceptible-infected-recovered-susceptible (SIRS) model. The emergence of the Omicron BA.1 variant was observed, replacing the presence of its predecessor, the Delta variant. Comparative analysis between the wastewater data and the modified SIRS model effectively described the BA.1 and subsequent BA.2 waves, with the decline of the Delta variant aligning with its diminished presence below the detection threshold in wastewater. This study demonstrates the potential of WBE as a valuable tool for future pandemics. Furthermore, by analyzing the sensitivity of different variants to model parameters, we are able to deduce real-life values of cross-variant immunity probabilities, emphasizing the asymmetry in their strength.

3,3'-Diindolylmethane (DIM): A Potential Therapeutic Agent against Cariogenic Streptococcus mutans Biofilm.

Indole, a metabolite of the amino acid tryptophan, has been proven to act as a signal molecule in bacteria, acting in different aspects of biofilm formation. The oral biofilm is a type of biofilm that has consequences for human health. It is a complex, three-dimensional structure that develops on the surface of teeth via the attachment of primary microbial colonizers. Many oral infections are caused by an imbalance occurring in the microorganisms naturally found in oral biofilms and are considered major public health concerns. In this study, we test the effect of a natural bis-indole, 3,3'-Diindolylmethane (DIM), in mitigating the pathogenicity of the oral biofilm inhabiting bacterium Streptococcus mutans, a bacterium that is considered to be a principal etiological agent in dental caries. Our study found that DIM was able to attenuate S. mutans biofilm formation by 92%. Additionally, treatment with DIM lowered extracellular polymeric substance (EPS) production and decreased its durability significantly under acidic conditions. Therefore, the anti-biofilm and anti-virulence properties of DIM against S. mutans bacteria in an "oral setting" provides evidence for its usefulness in reducing biofilm formation and potentially for caries attenuation.

Spatial Succession Underlies Microbial Contribution to Food Digestion in the Gut of an Algivorous Sea Urchin.

Dietary influence on the microbiome in algivorous sea urchins such as Tripneustes gratilla elatensis suggests a bacterial contribution to the digestion of fiber-rich seaweed. An ecological insight into the spatial arrangement in the gut bacterial community will improve our knowledge of host-microbe relations concerning the involved taxa, their metabolic repertoire, and the niches of activity. Toward this goal, we investigated the bacterial communities in the esophagus, stomach, and intestine of Ulva-fed sea urchins through 16S rRNA amplicon sequencing, followed by the prediction of their functional genes. We revealed communities with distinct features, especially those in the esophagus and intestine. The esophageal community was less diverse and was poor in food digestive or fermentation genes. In contrast, bacteria that can contribute to the digestion of the dietary Ulva were common in the stomach and intestine and consisted of genes for carbohydrate decomposition, fermentation, synthesis of short-chain fatty acids, and various ways of N and S metabolism. Bacteroidetes and Firmicutes were found as the main phyla in the gut and are presumably also necessary in food digestion. The abundant sulfate-reducing bacteria in the stomach and intestine from the genera Desulfotalea, Desulfitispora, and Defluviitalea may aid in removing the excess sulfate from the decomposition of the algal polysaccharides. Although these sea urchins were fed with Ulva, genes for the degradation of polysaccharides of other algae and plants were present in this sea urchin gut microbiome. We conclude that the succession of microbial communities along the gut obtained supports the hypothesis on bacterial contribution to food digestion. IMPORTANCE Alga grazing by the sea urchin Tripneustes gratilla elatensis is vital for nutrient recycling and constructing new reefs. This research was driven by the need to expand the knowledge of bacteria that may aid this host in alga digestion and their phylogeny, roles, and activity niches. We hypothesized alterations in the bacterial compositional structure along the gut and their association with the potential contribution to food digestion. The current spatial insight into the sea urchin's gut microbiome ecology is novel and reveals how distinct bacterial communities are when distant from each other in this organ. It points to keynote bacteria with genes that may aid the host in the digestion of the complex sulfated polysaccharides in dietary Ulva by removing the released sulfates and fermentation to provide energy. The gut bacteria's genomic arsenal may also help to gain energy from diets of other algae and plants.

Biofilm growth monitoring using guided wave ultralong-range Surface Plasmon Resonance: A proof of concept.

Unwelcomed biofilms are problematic in food industries, surgical devices, marine applications, and wastewater treatment plants, essentially everywhere where there is moisture. Very recently, label-free advanced sensors such as localized and extended surface plasmon resonance (SPR) have been explored as tools for monitoring biofilm formation. However, conventional noble metal SPR substrates suffer from low penetration depth (100-300 nm) into the dielectric medium above the surface, preventing the reliable detection of large entities of single or multi-layered cell assemblies like biofilms which can grow up to a few micrometers or more. In this study, we propose using a plasmonic insulator-metal-insulator (IMI) structure (SiO2-Ag-SiO2) with a higher penetration depth based on a diverging beam single wavelength format of Kretschmann configuration in a portable SPR device. An SPR line detection algorithm for locating the reflectance minimum of the device helps to view changes in refractive index and accumulation of the biofilm in real-time down to 10-7 RIU precision. The optimized IMI structure exhibits strong penetration dependence on wavelength and incidence angle. Within the plasmonic resonance, different angles penetrate different depths, showing a maximum near the critical angle. At the wavelength of 635 nm, a high penetration depth of more than 4 µm was obtained. Compared to a thin gold film substrate, for which the penetration depth is only ∼200 nm, the IMI substrate provides more reliable results. The average thickness of the biofilm after 24 h of growth was found to be between 6 and 7 µm with ∼63% live cell volume, as estimated from confocal microscopic images using an image processing tool. To explain this saturation thickness, a graded index biofilm structure is proposed in which the refractive index decreases with the distance from the interface. Furthermore, when plasma-assisted degeneration of biofilms was studied in a semi-real-time format, there was almost no effect on the IMI substrate compared to the gold substrate. The growth rate over the SiO2 surface was higher than on gold, possibly due to differences between surface charge effects. On the gold, the excited plasmon generates an oscillating cloud of electrons, while for the SiO2 case, this does not happen. This methodology can be utilized to detect and characterize biofilms with better signal reliability with respect to concentration and size dependence.

Conversations in the Gut: The Role of Quorum Sensing in Normobiosis.

An imbalance in gut microbiota, termed dysbiosis, has been shown to affect host health. Several factors, including dietary changes, have been reported to cause dysbiosis with its associated pathologies that include inflammatory bowel disease, cancer, obesity, depression, and autism. We recently demonstrated the inhibitory effects of artificial sweeteners on bacterial quorum sensing (QS) and proposed that QS inhibition may be one mechanism behind such dysbiosis. QS is a complex network of cell-cell communication that is mediated by small diffusible molecules known as autoinducers (AIs). Using AIs, bacteria interact with one another and coordinate their gene expression based on their population density for the benefit of the whole community or one group over another. Bacteria that cannot synthesize their own AIs secretly "listen" to the signals produced by other bacteria, a phenomenon known as "eavesdropping". AIs impact gut microbiota equilibrium by mediating intra- and interspecies interactions as well as interkingdom communication. In this review, we discuss the role of QS in normobiosis (the normal balance of bacteria in the gut) and how interference in QS causes gut microbial imbalance. First, we present a review of QS discovery and then highlight the various QS signaling molecules used by bacteria in the gut. We also explore strategies that promote gut bacterial activity via QS activation and provide prospects for the future.

Intrinsic and bioaugmented aerobic trichloroethene degradation at seven sites.

Trichloroethene (TCE) is one of the most prevalent contaminants in groundwater pollution worldwide. Aerobic-metabolic degradation of TCE has only recently been discovered at one field site. It has significant advantages over aerobic co-metabolism because no auxiliary substrates are required, and the oxygen demand is considerably lower. This study investigated the intrinsic degradation potential as well as the stimulation potential by bioaugmentation in microcosm experiments with groundwater from seven different sites contaminated with chloroethenes. An enrichment culture metabolizing TCE aerobically served as inoculum. The groundwater samples were inoculated with liquid culture in mineral salts medium as well as with immobilized culture on silica sand. Additionally, some samples were inoculated with groundwater from the site where the enrichment culture originated. The microcosms without inoculum proved the occurrence of aerobic TCE-metabolizing bacteria stimulated by the supply of oxygen in 54% of the groundwater samples. TCE degradation started in most cases after adaptation times of up to 92 d. The doubling time of 24 d indicated comparatively slow growth of the aerobic TCE degrading microorganisms. Bioaugmentation triggered or accelerated TCE-degradation in all microcosms with chlorothene concentrations below 100 mg L-1. All inoculation strategies (liquid and immobilized enrichment culture or addition of groundwater from the active field site) were successful. Our study demonstrates that aerobic-metabolic TCE degradation can occur and be stimulated across a broad hydrogeologic spectrum and should be considered as a viable option for groundwater remediation at TCE-contaminated sites.

Complexes of Cu-Polysaccharide of a Marine Red Microalga Produce Spikes with Antimicrobial Activity.

Metal-polysaccharides have recently raised significant interest due to their multifunctional bioactivities. The antimicrobial activity of a complex of Cu2O with the sulfated polysaccharide (PS) of the marine red microalga Porphyridium sp. was previously attributed to spikes formed on the complex surface (roughness). This hypothesis was further examined here using other Cu-PS complexes (i.e., monovalent-Cu2O, CuCl and divalent-CuO, CuCl2). The nanostructure parameters of the monovalent complexes, namely, longer spikes (1000 nm) and greater density (2000-5000 spikes/µm2) were found to be related to the superior inhibition of microbial growth and viability and biofilm formation. When Escherichia coli TV1061, used as a bioluminescent test organism, was exposed to the monovalent Cu-PS complexes, enhanced bioluminescence accumulation was observed, probably due to membrane perforation by the spikes on the surface of the complexes and consequent cytoplasmic leakage. In addition, differences were found in the surface chemistry of the monovalent and divalent Cu-PS complexes, with the monovalent Cu-PS complexes exhibiting greater stability (ζ-potential, FTIR spectra, and leaching out), which could be related to spike formation. This study thus supports our hypothesis that the spikes protruding from the monovalent Cu-PS surfaces, as characterized by their aspect ratio, are responsible for the antimicrobial and antibiofilm activities of the complexes.

The Microbiome Structure of the Symbiosis between the Desert Truffle Terfezia boudieri and Its Host Plant Helianthemum sessiliflorum.

The desert truffle Terfezia boudieri is an ascomycete fungus that forms ect-endomycorrhiza in the roots of plants belonging to Cistaceae. The fungus forms hypogeous edible fruit bodies, appreciated as gourmet food. Truffles and host plants are colonized by various microbes, which may contribute to their development. However, the diversity and composition of the bacterial community under field conditions in the Negev desert are still unknown. The overall goal of this research was to identify the rhizosphere microbial community supporting the establishment of a symbiotic association between T. boudieri and Helianthemum sessiliflorum. The bacterial community was characterized by fruiting bodies, mycorrhized roots, and rhizosphere soil. Based on next-generation sequencing meta-analyses of the 16S rRNA gene, we discovered diverse bacterial communities of fruit bodies that differed from those found in the roots and rhizosphere. Families of Proteobacteria, Planctomycetes, and Actinobacteria were present in all four samples. Alpha diversity analysis revealed that the rhizosphere and roots contain significantly higher bacterial species numbers compared to the fruit. Additionally, ANOSIM and PCoA provided a comparative analysis of the bacterial taxa associated with fruiting bodies, roots, and rhizosphere. The core microbiome described consists of groups whose biological role triggers important traits supporting plant growth and fruit body development.

Carbohydrate-Active Enzymes of a Novel Halotolerant Alkalihalobacillus Species for Hydrolysis of Starch and Other Algal Polysaccharides.

Halotolerant bacteria capable of starch hydrolysis by their amylases will benefit various industries, specifically since the hydrolytic activity of current industrial amylases is inhibited or even absent in salt-rich or alkaline environments. Seeking novel enzymes, we analyzed the entire genome content of a marine bacterium isolated from the gut of sea urchins to compare it against other bacterial genomes. Conditions underlying α-amylase activity were examined in vitro at various salinities (0 to 4%) and temperatures (25°C to 37°C). Genomic analyses revealed the isolated bacterium as a new species of Alkalihalobacillus. Comparative analysis of the contents of carbohydrate-active enzymes revealed various α-amylases, each with its respective carbohydrate-binding module for starch hydrolysis. Functional analysis identified the hydrolysis of starch and the maltooligosaccharides maltose and dextrin into d- and UDP-glucose. The fastest growth and α-amylase production occurred at 3% salinity at a temperature of 30°C. The Alkalihalobacillus sp. consists of exclusive contents of α-amylases and other enzymes that may be valuable in the hydrolysis of the algal polysaccharides cellulose and laminarin. IMPORTANCE Toward the discovery of novel carbohydrate-active enzymes that may be useful in the hydrolysis of starch, we examined a halotolerant bacterial isolate of Alkalihalobacillus sp. regarding its genomic content and conditions underlying the production of active α-amylases. The production of α-amylases was measured in bacterial cultures at relatively high temperature (37°C) and salinity (4%). The Alkalihalobacillus sp. revealed an exclusive content of amylases and other carbohydrate-active enzymes compared to other relevant bacteria. These enzymes may be valuable for the hydrolysis of algal polysaccharides. The enzymatic cascade of the Alkalihalobacillus sp. for starch metabolism allows polysaccharide degradation into monosugars while preventing the accumulation of intermediate inhibitors of maltose or dextrin.

Anti-Virulence Activity of 3,3'-Diindolylmethane (DIM): A Bioactive Cruciferous Phytochemical with Accelerated Wound Healing Benefits.

Antimicrobial resistance is among the top global health problems with antibacterial resistance currently representing the major threat both in terms of occurrence and complexity. One reason current treatments of bacterial diseases are ineffective is the occurrence of protective and resistant biofilm structures. Phytochemicals are currently being reviewed for newer anti-virulence agents. In the present study, we aimed to investigate the anti-virulence activity of 3,3'-diindolylmethane (DIM), a bioactive cruciferous phytochemical. Using a series of in vitro assays on major Gram-negative pathogens, including transcriptomic analysis, and in vivo porcine wound studies as well as in silico experiments, we show that DIM has anti-biofilm activity. Following DIM treatment, our findings show that biofilm formation of two of the most prioritized bacterial pathogens Acinetobacter baumannii and Pseudomonas aeruginosa was inhibited respectively by 65% and 70%. Combining the antibiotic tobramycin with DIM enabled a high inhibition (94%) of P. aeruginosa biofilm. A DIM-based formulation, evaluated for its wound-healing efficacy on P. aeruginosa-infected wounds, showed a reduction in its bacterial bioburden, and wound size. RNA-seq was used to evaluate the molecular mechanism underlying the bacterial response to DIM. The gene expression profile encompassed shifts in virulence and biofilm-associated genes. A network regulation analysis showed the downregulation of 14 virulence-associated super-regulators. Quantitative real-time PCR verified and supported the transcriptomic results. Molecular docking and interaction profiling indicate that DIM can be accommodated in the autoinducer- or DNA-binding pockets of the virulence regulators making multiple non-covalent interactions with the key residues that are involved in ligand binding. DIM treatment prevented biofilm formation and destroyed existing biofilm without affecting microbial death rates. This study provides evidence for bacterial virulence attenuation by DIM.

Managing an evolving pandemic: Cryptic circulation of the Delta variant during the Omicron rise.

SARS-CoV-2 continued circulation results in mutations and the emergence of various variants. Until now, whenever a new, dominant, variant appeared, it overpowered its predecessor after a short parallel period. The latest variant of concern, Omicron, is spreading swiftly around the world with record morbidity reports. Unlike the Delta variant, previously considered to be the main variant of concern in most countries, including Israel, the dynamics of the Omicron variant showed different characteristics. To enable quick assessment of the spread of this variant we developed an RT-qPCR primers-probe set for the direct detection of Omicron variant. Characterized as highly specific and sensitive, the new Omicron detection set was deployed on clinical and wastewater samples. In contrast to the expected dynamics whereupon the Delta variant diminishes as Omicron variant increases, representative results received from wastewater detection indicated a cryptic circulation of the Delta variant even with the increased levels of Omicron variant. Resulting wastewater data illustrated the very initial Delta-Omicron dynamics occurring in real time. Despite this, the future development and dynamics of the two variants side-by-side is still mainly unknown. Based on the initial results, a double susceptible-infected-recovered model was developed for the Delta and Omicron variants. According to the developed model, it can be expected that the Omicron levels will decrease until eliminated, while Delta variant will maintain its cryptic circulation. If this comes to pass, the mentioned cryptic circulation may result in the reemergence of a Delta morbidity wave or in the possible generation of a new threatening variant. In conclusion, the deployment of wastewater-based epidemiology is recommended as a convenient and representative tool for pandemic containment.

Probiotic Characteristics of Lactiplantibacillus Plantarum N-1 and Its Cholesterol-Lowering Effect in Hypercholesterolemic Rats.

In this study, the probiotic potential and treatment effects of Lactiplantibacillus plantarum N-1 in hypercholesterolemic rats were investigated, and the possible regulatory mechanisms of lipid metabolism via short-chain fatty acids (SCFAs) and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase were elucidated. The strain N-1 displayed probiotic properties of antioxidant capacity, adhesion to Caco-2 cells, susceptibility to antibiotics in vitro. The results in animal study showed that the total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels in serum and TC in liver declined significantly in both N-1 and simvastatin (Sta) treatment groups compared to the control (P < 0.05), and the extent of these decreases were similar between them. The expression of the HMG-CoA gene in the N-1 group was downregulated significantly by 31.18% compared to the control (P < 0.01), and the contents of butyrate and valerate in N-1 groups were significantly higher than those in both model and Sta group (P < 0.05). Thus, promoting the production of the intestinal SCFAs and inhibiting the expression of HMG-CoA reductase by L. plantarum N-1 may contribute to the improved lipid metabolism and thus lowering cholesterol level in rats. Our investigation indicated that L. plantarum N-1 has the potential to be developed into a functional food supplement for hypercholesterolemia treatment.

Microscopic and biomolecular complementary approaches to characterize bioweathering processes at petroglyph sites from the Negev Desert, Israel.

Throughout the Negev Desert highlands, thousands of ancient petroglyphs sites are susceptible to deterioration processes that may result in the loss of this unique rock art. Therefore, the overarching goal of the current study was to characterize the composition, diversity and effects of microbial colonization of the rocks to find ways of protecting these unique treasures. The spatial organization of the microbial colonizers and their relationships with the lithic substrate were analysed using scanning electron microscopy. This approach revealed extensive epilithic and endolithic colonization and close microbial-mineral interactions. Shotgun sequencing analysis revealed various taxa from the archaea, bacteria and some eukaryotes. Metagenomic coding sequences (CDS) of these microbial lithobionts exhibited specific metabolic pathways involved in the rock elements' cycles and uptake processes. Thus, our results provide evidence for the potential participation of the microorganisms colonizing these rocks during different solubilization and mineralization processes. These damaging actions may contribute to the deterioration of this extraordinary rock art and thus threaten this valuable heritage. Shotgun metagenomic sequencing, in conjunction with the in situ scanning electron microscopy study, can thus be considered an effective strategy to understand the complexity of the weathering processes occurring at petroglyph sites and other cultural heritage assets.

Mono-specific algal diets shape microbial networking in the gut of the sea urchin Tripneustes gratilla elatensis.

BACKGROUND: Algivorous sea urchins can obtain energy from a diet of a single algal species, which may result in consequent changes in their gut microbe assemblies and association networks. METHODS: To ascertain whether such changes are led by specific microbes or limited to a specific region in the gut, we compared the microbial assembly in the three major gut regions of the sea urchin Tripneustes gratilla elatensis when fed a mono-specific algal diet of either Ulva fasciata or Gracilaria conferta, or an algal-free diet. DNA extracts from 5 to 7 individuals from each diet treatment were used for Illumina MiSeq based 16S rRNA gene sequencing (V3-V4 region). Niche breadth of each microbe in the assembly was calculated for identification of core, generalist, specialist, or unique microbes. Network analyzers were used to measure the connectivity of the entire assembly and of each of the microbes within it and whether it altered with a given diet or gut region. Lastly, the predicted metabolic functions of key microbes in the gut were analyzed to evaluate their potential contribution to decomposition of dietary algal polysaccharides. RESULTS: Sea urchins fed with U. fasciata grew faster and their gut microbiome network was rich in bacterial associations (edges) and networking clusters. Bacteroidetes was the keystone microbe phylum in the gut, with core, generalist, and specialist representatives. A few microbes of this phylum were central hub nodes that maintained community connectivity, while others were driver microbes that led the rewiring of the assembly network based on diet type through changes in their associations and centrality. Niche breadth agreed with microbes' richness in genes for carbohydrate active enzymes and correlated Bacteroidetes specialists to decomposition of specific polysaccharides in the algal diets. CONCLUSIONS: The dense and well-connected microbial network in the gut of Ulva-fed sea urchins, together with animal's rapid growth, may suggest that this alga was most nutritious among the experimental diets. Our findings expand the knowledge on the gut microbial assembly in T. gratilla elatensis and strengthen the correlation between microbes' generalism or specialism in terms of occurrence in different niches and their metabolic arsenal which may aid host nutrition.

RT-qPCR assays for SARS-CoV-2 variants of concern in wastewater reveals compromised vaccination-induced immunity.

SARS-CoV-2 variants of concern, demonstrating higher infection rate and lower vaccine effectiveness as compared with the original virus, are important factors propelling the ongoing COVID-19 global outbreak. Therefore, prompt identification of these variants in the environment is essential for pandemic assessment and containment efforts. One well established tool for such viral monitoring is the use of wastewater systems. Here, we describe continuous monitoring of traces of SARS-CoV-2 viruses in the municipal wastewater of a large city in Israel. By observing morbidity fluctuations (during three main COVID-19 surges) occurring in parallel with Pfizer-BioNTech COVID-19 vaccine vaccination rate, compromised immunity was revealed in the current morbidity peak. RT-qPCR assays for the Original (D614G), Alpha and Beta variants had been previously developed and are being employed for wastewater surveillance. In the present study we developed a sensitive RT-qPCR assay designed for the rapid, direct detection of Gamma and Delta variants of concern. Sensitive quantification and detection of the various variants showed the prevalence of the original variant during the first morbidity peak. The dominance of the Alpha variant over the original variant correlated with the second morbidity peak. These variants decreased concurrently with an increase in vaccinations (Feb-March 2021) and the observed decrease in morbidity. The appearance and subsequent rise of the Delta variant became evident and corresponded to the third morbidity peak (June-August 2021). These results suggest a high vaccine neutralization efficiency towards the Alpha variant compared to its neutralization efficiency towards the Delta variant. Moreover, the third vaccination dose (booster) seems to regain neutralization efficiency towards the Delta variant. The developed assays and wastewater-based epidemiology are important tools aiding in morbidity surveillance and disclosing vaccination efforts and immunity dynamics in the community.

Inhibitory Effects of Artificial Sweeteners on Bacterial Quorum Sensing.

Despite having been tagged as safe and beneficial, recent evidence remains inconclusive regarding the status of artificial sweeteners and their putative effects on gut microbiota. Gut microorganisms are essential for the normal metabolic functions of their host. These microorganisms communicate within their community and regulate group behaviors via a molecular system termed quorum sensing (QS). In the present study, we aimed to study the effects of artificial sweeteners on this bacterial communication system. Using biosensor assays, biophysical protein characterization methods, microscale thermophoresis, swarming motility assays, growth assays, as well as molecular docking, we show that aspartame, sucralose, and saccharin have significant inhibitory actions on the Gram-negative bacteria N-acyl homoserine lactone-based (AHL) communication system. Our studies indicate that these three artificial sweeteners are not bactericidal. Protein-ligand docking and interaction profiling, using LasR as a representative participating receptor for AHL, suggest that the artificial sweeteners bind to the ligand-binding pocket of the protein, possibly interfering with the proper housing of the native ligand and thus impeding protein folding. Our findings suggest that these artificial sweeteners may affect the balance of the gut microbial community via QS-inhibition. We, therefore, infer an effect of these artificial sweeteners on numerous molecular events that are at the core of intestinal microbial function, and by extension on the host metabolism.

City-level SARS-CoV-2 sewage surveillance.

The COVID-19 pandemic created a global crisis impacting not only healthcare systems, but also economics and society. Therefore, it is important to find novel methods for monitoring disease activity. Recent data have indicated that fecal shedding of SARS-CoV-2 is common, and that viral RNA can be detected in wastewater. This suggests that wastewater monitoring is a potentially efficient tool for both epidemiological surveillance, and early warning for SARS-CoV-2 circulation at the population level. In this study we sampled an urban wastewater infrastructure in the city of Ashkelon (Ì´ 150,000 population), Israel, during the end of the first COVID-19 wave in May 2020 when the number of infections seemed to be waning. We were able to show varying presence of SARS-CoV-2 RNA in wastewater from several locations in the city during two sampling periods, before the resurgence was clinically apparent. This was expressed with a new index, Normalized Viral Load (NVL) which can be used in different area scales to define levels of virus activity such as red (high) or green (no), and to follow morbidity in the population at the tested area. The rise in viral load between the two sampling periods (one week apart) indicated an increase in morbidity that was evident two weeks to a month later in the population. Thus, this methodology may provide an early indication for SARS-CoV-2 infection outbreak in a population before an outbreak is clinically apparent.

Direct RT-qPCR assay for SARS-CoV-2 variants of concern (Alpha, B.1.1.7 and Beta, B.1.351) detection and quantification in wastewater.

Less than a year following the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak, variants of concern have emerged in the form of variant Alpha (B.1.1.7, the British variant) and Beta (B.1.351, the South Africa variant). Due to their high infectivity and morbidity, it has become clear that it is crucial to quickly and effectively detect these and other variants. Here, we report improved primers-probe sets for reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) for SARS-CoV-2 detection including a rapid, cost-effective, and direct RT-qPCR method for detection of the two variants of concern (Alpha, B.1.1.7 and Beta, B.1.351). All the developed primers-probe sets were fully characterized, demonstrating sensitive and specific detection. These primer-probe sets were also successfully employed on wastewater samples aimed at detecting and even quantifying new variants in a geographical area, even prior to the reports by the medical testing. The novel primers-probe sets presented here will enable proper responses for pandemic containment, particularly considering the emergence of variants of concern.