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.

Quorum Sensing and NF-κB Inhibition of Synthetic Coumaperine Derivatives from Piper nigrum.

Bacterial communication, termed Quorum Sensing (QS), is a promising target for virulence attenuation and the treatment of bacterial infections. Infections cause inflammation, a process regulated by a number of cellular factors, including the transcription Nuclear Factor kappa B (NF-κB); this factor is found to be upregulated in many inflammatory diseases, including those induced by bacterial infection. In this study, we tested 32 synthetic derivatives of coumaperine (CP), a known natural compound found in pepper (Piper nigrum), for Quorum Sensing Inhibition (QSI) and NF-κB inhibitory activities. Of the compounds tested, seven were found to have high QSI activity, three inhibited bacterial growth and five inhibited NF-κB. In addition, some of the CP compounds were active in more than one test. For example, compounds CP-286, CP-215 and CP-158 were not cytotoxic, inhibited NF-κB activation and QS but did not show antibacterial activity. CP-154 inhibited QS, decreased NF-κB activation and inhibited bacterial growth. Our results indicate that these synthetic molecules may provide a basis for further development of novel therapeutic agents against bacterial infections.

Functional marine metagenomic screening for anti-quorum sensing and anti-biofilm activity.

Quorum sensing (QS), a cell-to-cell communication process, entails the production of signaling molecules that enable synchronized gene expression in microbial communities to regulate myriad microbial functions, including biofilm formation. QS disruption may constitute an innovative approach to the design of novel antifouling and anti-biofilm agents. To identify novel quorum sensing inhibitors (QSI), 2,500 environmental bacterial artificial chromosomes (BAC) from uncultured marine planktonic bacteria were screened for QSI activity using soft agar overlaid with wild type Chromobacterium violaceum as an indicator. Of the BAC library clones, 7% showed high QSI activity (>40%) against the indicator bacterium, suggesting that QSI is common in the marine environment. The most active compound, eluted from BAC clone 14-A5, disrupted QS signaling pathways and reduced biofilm formation in both Pseudomonas aeruginosa and Acinetobacter baumannii. The mass spectra of the active BAC clone (14-A5) that had been visualized by thin layer chromatography was dominated by a m/z peak of 362.1.

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.

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.

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.

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.

Cross-kingdom inhibition of bacterial virulence and communication by probiotic yeast metabolites.

BACKGROUND: Probiotic milk-fermented microorganism mixtures (e.g., yogurt, kefir) are perceived as contributing to human health, and possibly capable of protecting against bacterial infections. Co-existence of probiotic microorganisms are likely maintained via complex biomolecular mechanisms, secreted metabolites mediating cell-cell communication, and other yet-unknown biochemical pathways. In particular, deciphering molecular mechanisms by which probiotic microorganisms inhibit proliferation of pathogenic bacteria would be highly important for understanding both the potential benefits of probiotic foods as well as maintenance of healthy gut microbiome. RESULTS: The microbiome of a unique milk-fermented microorganism mixture was determined, revealing a predominance of the fungus Kluyveromyces marxianus. We further identified a new fungus-secreted metabolite-tryptophol acetate-which inhibits bacterial communication and virulence. We discovered that tryptophol acetate blocks quorum sensing (QS) of several Gram-negative bacteria, particularly Vibrio cholerae, a prominent gut pathogen. Notably, this is the first report of tryptophol acetate production by a yeast and role of the molecule as a signaling agent. Furthermore, mechanisms underscoring the anti-QS and anti-virulence activities of tryptophol acetate were elucidated, specifically down- or upregulation of distinct genes associated with V. cholerae QS and virulence pathways. CONCLUSIONS: This study illuminates a yet-unrecognized mechanism for cross-kingdom inhibition of pathogenic bacteria cell-cell communication in a probiotic microorganism mixture. A newly identified fungus-secreted molecule-tryptophol acetate-was shown to disrupt quorum sensing pathways of the human gut pathogen V. cholerae. Cross-kingdom interference in quorum sensing may play important roles in enabling microorganism co-existence in multi-population environments, such as probiotic foods and the gut microbiome. This discovery may account for anti-virulence properties of the human microbiome and could aid elucidating health benefits of probiotic products against bacterially associated diseases. Video Abstract.

Sniffing Bacteria with a Carbon-Dot Artificial Nose.

HIGHLIGHTS: Novel artificial nose based upon electrode-deposited carbon dots (C-dots). Significant selectivity and sensitivity determined by "polarity matching" between the C-dots and gas molecules. The C-dot artificial nose facilitates, for the first time, real-time, continuous monitoring of bacterial proliferation and discrimination among bacterial species, both between Gram-positive and Gram-negative bacteria and between specific strains. Machine learning algorithm furnishes excellent predictability both in the case of individual gases and for complex gas mixtures. Continuous, real-time monitoring and identification of bacteria through detection of microbially emitted volatile molecules are highly sought albeit elusive goals. We introduce an artificial nose for sensing and distinguishing vapor molecules, based upon recording the capacitance of interdigitated electrodes (IDEs) coated with carbon dots (C-dots) exhibiting different polarities. Exposure of the C-dot-IDEs to volatile molecules induced rapid capacitance changes that were intimately dependent upon the polarities of both gas molecules and the electrode-deposited C-dots. We deciphered the mechanism of capacitance transformations, specifically substitution of electrode-adsorbed water by gas molecules, with concomitant changes in capacitance related to both the polarity and dielectric constants of the vapor molecules tested. The C-dot-IDE gas sensor exhibited excellent selectivity, aided by application of machine learning algorithms. The capacitive C-dot-IDE sensor was employed to continuously monitor microbial proliferation, discriminating among bacteria through detection of distinctive "volatile compound fingerprint" for each bacterial species. The C-dot-IDE platform is robust, reusable, readily assembled from inexpensive building blocks and constitutes a versatile and powerful vehicle for gas sensing in general, bacterial monitoring in particular.

An inside look at a biofilm: Pseudomonas aeruginosa flagella biotracking.

The opportunistic pathogen, Pseudomonas aeruginosa, a flagellated bacterium, is one of the top model organisms for biofilm studies. To elucidate the location of bacterial flagella throughout the biofilm life cycle, we developed a new flagella biotracking tool. Bacterial flagella were site-specifically labeled via genetic code expansion. This enabled us to track bacterial flagella during biofilm maturation. Live flagella imaging revealed the presence and synthesis of flagella throughout the biofilm life cycle. To study the possible role of flagella in a biofilm, we produced a flagella knockout strain and compared its biofilm to that of the wild-type strain. Results showed a one order of magnitude stronger biofilm structure in the wild type in comparison with the flagella knockout strain. This suggests a possible structural role for flagella in a biofilm, conceivably as a scaffold. Our findings suggest a new model for biofilm maturation dynamic which underscores the importance of direct evidence from within the biofilm.

Tracking SARS-CoV-2 RNA through the Wastewater Treatment Process.

Municipal sewage carries degraded and intact viral particles and RNA (ribonucleic acid) of SARS-CoV-2 (severe acute respiratory coronavirus 2), shed by COVID-19 (coronavirus disease 2019) patients, to sewage and eventually to wastewater treatment plants. Proper wastewater treatment can prevent uncontrolled discharges of the virus into the environment. However, the role of different wastewater treatment stages in reducing viral RNA concentrations is, thus far, unknown. Here, we quantified SARS-CoV-2 RNA in raw sewage and during the main stages of the activated sludge process from two wastewater treatment plants in Israel, on three different days during the 2020 COVID-19 outbreak. To reduce the detection limit, samples were concentrated prior to quantification by real-time polymerase chain reaction by a factor of 2-43 using ultrafiltration. On average, ∼1 log RNA removal was attained by each of the primary and secondary treatment steps; however, >100 copies of SARS-CoV-2 RNA/mL remained in the secondary effluents. Following chlorination, SARS-CoV-2 RNA was detected only once, likely due to an insufficient chlorine dose. Our results emphasize the capabilities and limitations of the conventional wastewater treatment process in reducing the SARS-CoV-2 RNA concentration and present preliminary evidence for the importance of tertiary treatment and chlorination in reducing dissemination of the virus to the environment.

Regressing SARS-CoV-2 Sewage Measurements Onto COVID-19 Burden in the Population: A Proof-of-Concept for Quantitative Environmental Surveillance.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an RNA virus, a member of the coronavirus family of respiratory viruses that includes severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and the Middle East respiratory syndrome (MERS). It has had an acute and dramatic impact on health care systems, economies, and societies of affected countries during the past 8 months. Widespread testing and tracing efforts are being employed in many countries in attempts to contain and mitigate this pandemic. Recent data has indicated that fecal shedding of SARS-CoV-2 is common and that the virus RNA can be detected in wastewater. This indicates that wastewater monitoring may provide a potentially efficient tool for the epidemiological surveillance of SARS-CoV-2 infection in large populations at relevant scales. In particular, this provides important means of (i) estimating the extent of outbreaks and their spatial distributions, based primarily on in-sewer measurements, (ii) managing the early-warning system quantitatively and efficiently, and (iii) verifying disease elimination. Here we report different virus concentration methods using polyethylene glycol (PEG), alum, or filtration techniques as well as different RNA extraction methodologies, providing important insights regarding the detection of SARS-CoV-2 RNA in sewage. Virus RNA particles were detected in wastewater in several geographic locations in Israel. In addition, a correlation of virus RNA concentration to morbidity was detected in Bnei-Barak city during April 2020. This study presents a proof of concept for the use of direct raw sewage-associated virus data, during the pandemic in the country as a potential epidemiological tool.

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.