Identification of environmental and methodological factors driving variability of Pepper Mild Mottle Virus (PMMoV) across three wastewater treatment plants in the City of Toronto.

PMMoV has been widely used to normalize the concentration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, influenza, and respiratory syncytial virus (RSV) to account for variations in the fecal content of wastewater. PMMoV is also used as an internal RNA recovery control for wastewater-based epidemiology (WBE) tests. While potentially useful for the interpretation of WBE data, previous studies have suggested that PMMoV concentration can be affected by various physico-chemical characteristics of wastewater. There is also the possibility that laboratory methods, particularly the variability in centrifugation steps to remove supernatant from pellets can cause PMMoV variability. The goal of this study is to improve our understanding of the main drivers of PMMoV variability by assessing the relationship between PMMoV concentration, the physico-chemical characteristics of wastewater, and the methodological approach for concentrating wastewater samples. We analyzed 24-hour composite wastewater samples collected from the influent stream of three wastewater treatment plants (WWTPs) located in the City of Toronto, Ontario, Canada. Samples were collected 3 to 5 times per week starting from the beginning of March 2021 to mid-July 2023. The influent flow rate was used to partition the data into wet and dry weather conditions. Physico-chemical characteristics (e.g., total suspended solids (TSS), biological oxygen demand (BOD), alkalinity, electrical conductivity (EC), and ammonia (NH3)) of the raw wastewater were measured, and PMMoV was quantified. Spatial and temporal variability of PMMoV was observed throughout the study period. PMMoV concentration was significantly higher during dry weather conditions. Multiple linear regression analysis demonstrates that the number and type of physico-chemical parameters that drive PMMoV variability are site-specific, but overall BOD and alkalinity were the most important predictors. Differences in PMMoV concentration for a single WWTP between two different laboratory methods, along with a weak correlation between pellet mass and TSS using one method may indicate that differences in sample concentration and subjective subsampling bias could alter viral recovery and introduce variability to the data.

Probiotic Yeast Saccharomyces: Back to Nature to Improve Human Health.

Saccharomyces cerevisiae var. boulardii is best known for its treatment efficacy against different gastrointestinal diseases. This probiotic yeast can significantly protect the normal microbiota of the human gut and inhibit the pathogenicity of different diarrheal infections. Several clinical investigations have declared S. cerevisiae var. boulardii a biotherapeutic agent due to its antibacterial, antiviral, anti-carcinogenic, antioxidant, anti-inflammatory and immune-modulatory properties. Oral or intramuscular administration of S. cerevisiae var. boulardii can remarkably induce health-promoting effects in the host body. Different intrinsic and extrinsic factors are responsible for its efficacy against acute and chronic gut-associated diseases. This review will discuss the clinical and beneficial effects of S. cerevisiae var. boulardii in the treatment and prevention of different metabolic diseases and highlight some of its health-promising properties. This review article will provide fundamental insights for new avenues in the fields of biotherapeutics, antimicrobial resistance and one health.

Unveiling the Potential Role of Nanozymes in Combating the COVID-19 Outbreak.

The current coronavirus disease 2019 (COVID-19) outbreak is considered as one of the biggest public health challenges and medical emergencies of the century. A global health emergency demands an urgent development of rapid diagnostic tools and advanced therapeutics for the mitigation of COVID-19. To cope with the current crisis, nanotechnology offers a number of approaches based on abundance and versatile functioning. Despite major developments in early diagnostics and control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is still a need to find effective nanomaterials with low cost, high stability and easy use. Nanozymes are nanomaterials with innate enzyme-like characteristics and exhibit great potential for various biomedical applications such as disease diagnosis and anti-viral agents. Overall the potential and contribution of nanozymes in the fight against SARS-CoV-2 infection i.e., rapid detection, inhibition of the virus at various stages, and effective vaccine development strategies, is not fully explored. This paper discusses the utility and potential of nanozymes from the perspective of COVID-19. Moreover, future research directions and potential applications of nanozymes are highlighted to overcome the challenges related to early diagnosis and therapeutics development for the SARS-CoV-2. We anticipate the current perspective will play an effective role in the existing response to the COVID-19 crisis.

Turning harmful algal biomass to electricity by microbial fuel cell: A sustainable approach for waste management.

Effective utilization of harmful algal biomass from eutrophic lakes is required for sustainable waste management and circular bioeconomy. In this study, Microcystis aeruginosa derived biomass served as an electron donor in the microbial fuel cell (MFC) for waste treatment and electricity generation. Bioelectrochemical performance of MFC fed with microalgae (MFC-Algae) was compared with MFC fed with a commercial substrate (MFC-Acetate). Complete removal of microcystin-LR (MC-LR) and high chemical oxygen demand (COD) removal efficiency (67.5 ± 1%) in MFC-Algae showed that harmful algal biomass could be converted into bioelectricity. Polarization curves revealed that MFC-Algae delivered the maximum power density (83 mW/m2) and current density (672 mA/m2), which was 43% and 45% higher than that of MFC-Acetate respectively. Improved electrochemical performance and substantial coulombic efficiency (7.6%) also verified the potential use of harmful algal biomass as an alternate MFC substrate. Diverse microbial community profiles showed the substrate-dependent electrogenic activities in each MFC. Biodegradation pathway of MC-LR by anodic microbes was also explored in detail. Briefly, a sustainable approach for on-site waste management of harmful algal biomass was presented, which was deprived of transportation and special pretreatments. It is anticipated that current findings will help to pave the way for practical applications of MFC technology.

Establishing the relationship between molecular biomarkers and biotransformation rates: Extension of knowledge for dechlorination of polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs).

Anaerobic reductive treatment technologies offer cost-effective and large-scale treatment of chlorinated compounds, including polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs). The information about the degradation rates of these compounds in natural settings is critical but difficult to obtain because of slow degradation processes. Establishing a relationship between biotransformation rate and abundance of biomarkers is one of the most critical challenges faced by the bioremediation industry. When solved for a given contaminant, it may result in significant cost savings because of serving as a basis for action. In the current review, we have summarized the studies highlighting the use of biomarkers, particularly DNA and RNA, as a proxy for reductive dechlorination of chlorinated ethenes. As the use of biomarkers for predicting biotransformation rates has not yet been executed for PCDD/Fs, we propose the extension of the same knowledge for dioxins, where slow degradation rates further necessitate the need for developing the biomarker-rate relationship. For this, we have first retrieved and calculated the bioremediation rates of different PCDD/Fs and then highlighted the key sequences that can be used as potential biomarkers. We have also discussed the implications and hurdles in developing such a relationship. Improvements in current techniques and collaboration with some other fields, such as biokinetic modeling, can improve the predictive capability of the biomarkers so that they can be used for effectively predicting biotransformation rates of dioxins and related compounds. In the future, a valid and established relationship between biomarkers and biotransformation rates of dioxin may result in significant cost savings, whilst also serving as a basis for action.

Assessment of knowledge and attitude trends towards antimicrobial resistance (AMR) among the community members, pharmacists/pharmacy owners and physicians in district Sialkot, Pakistan.

Background: Antimicrobial resistance (AMR) is an emerging threat to public health worldwide. A significant evidence has suggested that the knowledge and attitude trends among the community, pharmacists and physicians can play a critical role in managing the ever increasing threat of AMR. Methods: A cross-sectional survey was performed using three specific self-administered questionnaires for community members, pharmacists/pharmacy owners and physicians on a randomly selected sample population of 473, 424 and 308 respectively. Bivariate and multivariate logistic regression and Pearson chi-square tests were performed during data analysis. Result: A response rate of 81.2% (n = 385), 37.7% (n = 160) and 53.9% (n = 166) was achieved for general community, pharmacists/pharmacy owners and physicians respectively. More than half of the community participants (55.6%; n = 214) possess poor knowledge of AMR. Furthermore, knowledge and attitude of the community participants were also found to be significantly correlated (r2 = 0.02) with each other. In 90.6% (n = 145) of the pharmacies included in the survey, a qualified pharmacist was not present at the time of the operations. Only 36.9% physicians (n = 60) knew about the environmental route of dissemination of AMR. Majority of the physicians agreed that AMR is a global problem and also recognize the need for initiating AMR stewardship programs. Conclusion: Our study will provide effective assessment and potential insights in designing tri-faceted interventions for rationalizing antibiotics consumption thus controlling the development and dissemination of AMR.

Revisiting the mechanistic pathways for bacterial mediated synthesis of noble metal nanoparticles.

Synthesis and application of reliable nanoscale materials is a progressive domain and the limelight of modern nanotechnology. Conventional physicochemical approaches for the synthesis of metal nanoparticles have become obsolete owing to costly and hazardous materials. There is a need to explore alternative, cost-effective and eco-friendly strategies for fabrication of nanoparticle (NPs). Green synthesis of noble metal nanoparticles has emerged as a promising approach in the last decade. Elucidation of the molecular mechanism is highly essential in the biological synthesis of noble metal nanoparticles (NPs) for the controlled size, shape, and monodispersity. Moreover, mechanistic insights will help to scale up the facile synthesis protocols and will enable biotransformation of toxic heavy metals hence also providing the detoxification effects. Therefore, the current review article has primarily targeted the mechanisms involved in the green synthesis of metal NPs, which have been reported during the last few years. Detailed mechanistic pathways have highlighted nitrate reductase as a principle reducing agent in the bacterial mediated synthesis and stabilization of NPs. Furthermore, we have highlighted the potential implications of these mechanisms in bioremediation and biomineralization processes, which can play a critical role in biogeochemical cycling and environmental impacts of heavy metals. We anticipate that this review article will help researchers to address the challenges of bioremediation and modern nanotechnology.

Contributions and Challenges of High Throughput qPCR for Determining Antimicrobial Resistance in the Environment: A Critical Review.

Expansion in whole genome sequencing and subsequent increase in antibiotic resistance targets have paved the way of high throughput qPCR (HT-qPCR) for analyzing hundreds of antimicrobial resistance genes (ARGs) in a single run. A meta-analysis of 51 selected studies is performed to evaluate ARGs abundance trends over the last 7 years. WaferGenTM SmartChip is found to be the most widely used HT-qPCR platform among others for evaluating ARGs. Up till now around 1000 environmental samples (excluding biological replicates) from different parts of the world have been analyzed on HT-qPCR. Calculated detection frequency and normalized ARGs abundance (ARGs/16S rRNA gene) reported in gut microbiome studies have shown a trend of low ARGs as compared to other environmental matrices. Disparities in the HT-qPCR data analysis which are causing difficulties to researchers in precise interpretation of results have been highlighted and a possible way forward for resolving them is also suggested. The potential of other amplification technologies and point of care or field deployable devices for analyzing ARGs have also been discussed in the review. Our review has focused on updated information regarding the role, current status and future perspectives of HT-qPCR in the field of antimicrobial resistance.

A Rare Case Series: Impacted Distomolars.

BACKGROUND: The occurrence of multiple supernumerary teeth in individuals without any associated syndrome is rare. Supernumerary teeth may occur in any region of the dental arch and are frequently observed in the maxillary region. But the occurrence of distomolars is rare, particularly mandibular distomolars are extremely rare. CASES PRESENTATION: In this paper, we present a series of case reports of maxillary and mandibular distomolars. CONCLUSION: The occurrence of distomolars is rare, but when detected patients should be kept under observation.

Antimicrobial Resistance in the Environment.

This review su mmarizes selected publications from 2017 highlighting the occurrence of antimicrobial resistance (AMR) genes in the environment with emphasis on the aquatic environment. The review also covers different treatment technologies being developed for AMR genes as an environmental contaminant. The progress made in the area of AMR gene databases and tools is also reviewed. Besides a brief introduction, the content is categorized into three main sections: i) Occurrence of AMR in the Environment, ii) Treatment technologies for AMR, and iii) AMR databases and tools.

Improved antifouling potential of polyether sulfone polymeric membrane containing silver nanoparticles: self-cleaning membranes.

A new strategy to enhance the antifouling potential of polyether sulfone (PES) membrane is presented. Chemically synthesized silver nanoparticles (AgNPs) were used to prepare a mixed-matrix PES membrane by the phase inversion technique. Primarily, AgNPs synthesis was confirmed by surface plasmon resonance at 410-430 nm using UV-Visible spectroscopy. X-ray diffraction analysis revealed that AgNPs were crystalline with a diameter of 21 ± 2 nm. Furthermore, PES membranes were characterized by energy dispersive X-ray spectroscopy to confirm the incorporation of AgNPs in membranes. Hydrophilicity of the membranes was enhanced, whereas roughness, mechanical strength and biofouling were relatively reduced after embedding the AgNPs. Antibacterial potential of AgNPs was evaluated for E. coli in the disc diffusion and colony-forming unit (CFU) count method. All of the membranes were assessed for antifouling activity by filtering a control dilution (106 CFU/ml) of E. coli and by counting CFU. Anti-biofouling activity of the membrane was observed with different concentrations of AgNPs. Maximum reduction (66%) was observed in membrane containing 1.5% of AgNPs. The addition of antibiotic ceftriaxone enhanced the antibacterial effect of AgNPs in PES membranes. Our practicable antifouling strategy may be applied to other polymeric membranes which may pave the new way to achieve sustainable and self-cleaning membrane reactors on large scale.

Antimicrobial Resistance in the Environment.

This review summarizes selected publications of 2016 with emphasis on occurrence and treatment of antibiotic resistance genes and bacteria in the aquatic environment and wastewater and drinking water treatment plants. The review is conducted with emphasis on fate, modeling, risk assessment and data analysis methodologies for characterizing abundance. After providing a brief introduction, the review is divided into the following four sections: i) Occurrence of AMR in the Environment, ii) Treatment Technologies for AMR, iii) Modeling of Fate, Risk, and Environmental Impact of AMR, and iv) ARG Databases and Pipelines.

TCDD administered on activated carbon eliminates bioavailability and subsequent shifts to a key murine gut commensal.

Activated carbon (AC) is an increasingly attractive remediation alternative for the sequestration of dioxins at contaminated sites globally. However, the potential for AC to reduce the bioavailability of dioxins in mammals and the residing gut microbiota has received less attention. This question was partially answered in a recent study examining 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced hallmark toxic responses in mice administered with TCDD sequestered by AC or freely available in corn oil by oral gavage. Results from that study support the use of AC to significantly reduce the bioavailability of TCDD to the host. Herein, we examined the bioavailability of TCDD sequestered to AC on a key murine gut commensal and the influence of AC on the community structure of the gut microbiota. The analysis included qPCR to quantify the expression of segmented filamentous bacteria (SFB) in the mouse ileum, which has responded to TCDD-induced host toxicity in previous studies and community structure via sequencing the 16S ribosomal RNA (rRNA) gene. The expression of SFB 16S rRNA gene and functional genes significantly increased with TCDD administered with corn oil vehicle. Such a response was absent when TCDD was sequestered by AC. In addition, AC appeared to have a minimal influence on murine gut community structure and diversity, affecting only the relative abundance of Lactobacillaceae and two other groups. Results of this study further support the remedial use of AC for eliminating bioavailability of TCDD to host and subsequent influence on the gut microbiome.

On-filter direct amplification of Legionella pneumophila for rapid assessment of its abundance and viability.

Guidelines and regulations to control Legionella pneumophila in cooling water systems of large buildings are evolving due to the increasing number of outbreaks. Rapid, on-site, simple, and sensitive quantification methods that are also able to assess viability may be extremely useful in monitoring and control. Culture-based methods for measuring L. pneumophila may take 4-10 days and qPCR-based methods are also slow, requiring at least a day from sample to result, albeit mainly due to the need for sample transport to a centralized laboratory. This study reports a rapid isothermal amplification method for L. pneumophila concentration and detection with live/dead differentiation under field conditions. Using an on-filter direct amplification (i.e., amplification of cells without DNA extraction and purification) approach with propidium monoazide (PMA), and a real time isothermal amplification platform (Gene-Z), L. pneumophila could be detected in 1-2 h at ∼1 cfu/100 ml of tap water. Signature sequences from 16S rRNA and cadA genes were used as genetic markers for L. pneumophila and loop-mediated isothermal amplification (LAMP) primers were designed using Primer Explorer V4. Result were also compared with direct amplification of cells spiked into distilled, tap, and cooling water samples as well as extracted DNA by qPCR. This method may be useful to managers of cooling water systems in large buildings for rapid detection of L. pneumophila. The overall approach of on-site sample concentration, on-filter amplification, and live/dead differentiation may be extended to other organisms where analytical sensitivity and speed are equally important.

Modeling Hybridization Kinetics of Gene Probes in a DNA Biochip Using FEMLAB.

Microfluidic DNA biochips capable of detecting specific DNA sequences are useful in medical diagnostics, drug discovery, food safety monitoring and agriculture. They are used as miniaturized platforms for analysis of nucleic acids-based biomarkers. Binding kinetics between immobilized single stranded DNA on the surface and its complementary strand present in the sample are of interest. To achieve optimal sensitivity with minimum sample size and rapid hybridization, ability to predict the kinetics of hybridization based on the thermodynamic characteristics of the probe is crucial. In this study, a computer aided numerical model for the design and optimization of a flow-through biochip was developed using a finite element technique packaged software tool (FEMLAB; package included in COMSOL Multiphysics) to simulate the transport of DNA through a microfluidic chamber to the reaction surface. The model accounts for fluid flow, convection and diffusion in the channel and on the reaction surface. Concentration, association rate constant, dissociation rate constant, recirculation flow rate, and temperature were key parameters affecting the rate of hybridization. The model predicted the kinetic profile and signal intensities of eighteen 20-mer probes targeting vancomycin resistance genes (VRGs). Predicted signal intensities and hybridization kinetics strongly correlated with experimental data in the biochip (R² = 0.8131).

Virulence factor activity relationships (VFARs): a bioinformatics perspective.

Virulence factor activity relationships (VFARs) - a concept loosely based on quantitative structure-activity relationships (QSARs) for chemicals was proposed as a predictive tool for ranking risks due to microorganisms relevant to water safety. A rapid increase in sequencing capabilities and bioinformatics tools has significantly increased the potential for VFAR-based analyses. This review summarizes more than 20 bioinformatics databases and tools, developed over the last decade, along with their virulence and antimicrobial resistance prediction capabilities. With the number of bacterial whole genome sequences exceeding 241 000 and metagenomic analysis projects exceeding 13 000 and the ability to add additional genome sequences for few hundred dollars, it is evident that further development of VFARs is not limited by the availability of information at least at the genomic level. However, additional information related to co-occurrence, treatment response, modulation of virulence due to environmental and other factors, and economic impact must be gathered and incorporated in a manner that also addresses the associated uncertainties. Of the bioinformatics tools, a majority are either designed exclusively for virulence/resistance determination or equipped with a dedicated module. The remaining have the potential to be employed for evaluating virulence. This review focusing broadly on omics technologies and tools supports the notion that these tools are now sufficiently developed to allow the application of VFAR approaches combined with additional engineering and economic analyses to rank and prioritize organisms important to a given niche. Knowledge gaps do exist but can be filled with focused experimental and theoretical analyses that were unimaginable a decade ago. Further developments should consider the integration of the measurement of activity, risk, and uncertainty to improve the current capabilities.

Most probable number - loop mediated isothermal amplification (MPN-LAMP) for quantifying waterborne pathogens in <25min.

We are reporting a most probable number approach integrated to loop mediated isothermal technique (MPN-LAMP) focusing on Gram-negative Escherichia coli and Gram-positive Enterococcus faecalis bacterial cells without nucleic acids extraction. LAMP assays for uidA from E. coli and gelE from E. faecalis were successfully performed directly on cells up to single digit concentration using a commercial real time PCR instrument. Threshold time values of LAMP assays of bacterial cells, heat treated bacterial cells (95°C for 5min), and their purified genomic DNA templates were similar, implying that amplification could be achieved directly from bacterial cells at 63°C. Viability of bacterial cells was confirmed by using propidium monoazide in a LAMP assay with E. faecalis. To check its functionality on a microfluidic platform, MPN-LAMP assays targeting <10CFU of bacteria were also translated onto polymeric microchips and monitored by a low-cost fluorescence imaging system. The overall system provided signal-to-noise (SNR) ratios up to 800, analytical sensitivity of <10CFU, and time to positivity of about 20min. MPN-LAMP assays were performed for cell concentrations in the range of 105CFU to <10CFU. MPN values from LAMP assays confirmed that the amplifications were from <10CFU. The method described here, applicable directly on cells at 63°C, eliminates the requirement of complex nucleic acids extraction steps, facilitating the development of sensitive, rapid, low-cost, and field-deployable systems. This rapid MPN-LAMP approach has the potential to replace conventional MPN method for waterborne pathogens.

Chromosome 18p11.2 harbors susceptibility marker: D18S452, for bipolar affective disorder.

AIM: The aim of our study was to investigate whether the tandem repeat polymorphism in D18S452 microsatellite marker at locus 18p11.2 is a risk factor of bipolar affective disorder (BPAD) in Kashmiri population. MATERIALS AND METHODS: The repeat polymorphism in D18S452 was evaluated by polymerase chain reaction (PCR) analysis of in 74 diagnosed BPAD patients and 74 controls subjects. RESULTS: Tandem repeat (300 bp*) allele frequency was found to be 1.35% in controls and 8.108% in cases. The tandem repeat (250 bp*) allele frequency was found to be in 91.89% in cases and 98.65% in controls. The 252 bp/252 bp genotype was found to be present in 89.18% of cases and 98.64% of controls, the 300 bp/300 bp genotype in 5.40% of cases and 1.35% of controls and the 252 bp/300 bp variant in 5.40% of cases and none among the controls. Although the proportion of patients homozygous for tandem repeat (300 bp/300 bp) was higher in cases than in controls, the difference was not statistically significant when 252 bp/252 bp genotype was taken as reference (odds ratio [OR]=4.4242; 95% confidence interval [CI] 0.4822-40.5924); P=0.1529). However, when the frequency of heterozygous genotype (252 bp/300 bp) was compared with 252 bp/252 bp statistical significance was observed (OR=8.0603; 95% CI 1.1112-58.4646; P=0.0383). CONCLUSION: This is the first study reporting a significant association between D18S452 maker with tandem repeat polymorphism in heterozygous condition (252 bp/300 bp) and the development of BPAD in Kashmiri population.