Temporal and spatial relationships of CrAssphage and enteric viral and bacterial pathogens in wastewater in North Carolina.

Enteric disease remains one of the most common concerns for public health, particularly when it results from human exposure to surface and recreational waters contaminated with wastewater. Characterizing the temporal and spatial variation of enteric pathogens prevalent in wastewater is critical to develop approaches to mitigate their distribution in the environment. In this study, we aim to characterize pathogen variability and test the applicability of the human-associated wastewater indicator crAssphage as an indicator of enteric viral and bacterial pathogens. We conducted weekly samplings for 14 months from four wastewater treatment plants in North Carolina, USA. Untreated wastewater samples were processed using hollow fiber ultrafiltration, followed by secondary concentration methods. Adenovirus, norovirus, enterovirus, Salmonella, Shiga toxin 2 (stx2), Campylobacter, and crAssphage were measured by quantitative polymerase chain reaction (qPCR) and reverse transcriptase (rt)-qPCR. Our results revealed significant correlations between crAssphage and human adenovirus, enterovirus, norovirus, Salmonella, and Campylobacter (p<0.01). Pathogens and crAssphage concentrations in untreated wastewater showed distinct seasonal patterns, with peak concentrations of crAssphage and viral pathogens in fall and winter, while bacterial pathogens showed peaked concentrations in either winter (Campylobacter), fall (Salmonella), or summer (stx2). This study enhances the understanding of crAssphage as an alternative molecular indicator for both bacterial and viral pathogens. The findings of this study can also inform microbial modeling efforts for the prediction of the impact of wastewater pathogens on surface waters due to increased flooding events and wastewater overflows associated with climate change.

Autophagy Regulation Using Multimodal Chlorin e6-Loaded Polysilsesquioxane Nanoparticles to Improve Photodynamic Therapy.

Photodynamic therapy (PDT) is a promising anticancer noninvasive technique that relies on the generation of reactive oxygen species (ROS). Unfortunately, PDT still has many limitations, including the resistance developed by cancer cells to the cytotoxic effect of ROS. Autophagy, which is a stress response mechanism, has been reported as a cellular pathway that reduces cell death following PDT. Recent studies have demonstrated that PDT in combination with other therapies can eliminate anticancer resistance. However, combination therapy is usually challenged by the differences in the pharmacokinetics of the drugs. Nanomaterials are excellent delivery systems for the efficient codelivery of two or more therapeutic agents. In this work, we report on the use of polysilsesquioxane (PSilQ) nanoparticles for the codelivery of chlorin-e6 (Ce6) and an autophagy inhibitor for early- or late-stage autophagy. Our results, obtained from a reactive oxygen species (ROS) generation assay and apoptosis and autophagy flux analyses, demonstrate that the reduced autophagy flux mediated by the combination approach afforded an increase in the phototherapeutic efficacy of Ce6-PSilQ nanoparticles. We envision that the promising results in the use of multimodal Ce6-PSilQ material as a codelivery system against cancer pave the way for its future application with other clinically relevant combinations.

Antiviral activity of nano-monocaprin against Phi6 as a surrogate for SARS-CoV-2 / Actividad antiviral de nano-monocaprina contra Phi6 como sustituto del SARS-CoV-2

The COVID-19 pandemic involving SARS-CoV-2 has raised interest in using antimicrobial lipid formulations to inhibit viral entry into their host cells or to inactivate them. Lipids are a part of the innate defense mechanism against pathogens. Here, we evaluated the use of nano-monocaprin (NMC) in inhibiting enveloped (phi6) and unenveloped (MS2) bacteriophages. NMC was prepared using the sonochemistry technique. Size and morphology analysis revealed the formation of ~ 8.4 ± 0.2-nm NMC as measured by dynamic light scattering. We compared the antiviral activity of NMC with molecular monocaprin (MMC) at 0.5 mM and 2 mM concentrations against phi6, which we used as a surrogate for SARS-CoV-2. The synthesized NMC exhibited 50% higher antiviral activity against phi6 than MMC at pH 7 using plaque assay. NMC inactivated phi6 stronger at pH 4 than at pH 7. To determine if NMC is toxic to mammalian cells, we used MTS assay to assess its IC50 for HPDE and HeLa cell lines, which were ~ 203 and 221 µM, respectively. NMC may be used for prophylactic application either as a drop or spray since many viruses enter the human body through the mucosal lining of the nose, eyes, and lungs.(AU)

Antiviral activity of nano-monocaprin against Phi6 as a surrogate for SARS-CoV-2.

The COVID-19 pandemic involving SARS-CoV-2 has raised interest in using antimicrobial lipid formulations to inhibit viral entry into their host cells or to inactivate them. Lipids are a part of the innate defense mechanism against pathogens. Here, we evaluated the use of nano-monocaprin (NMC) in inhibiting enveloped (phi6) and unenveloped (MS2) bacteriophages. NMC was prepared using the sonochemistry technique. Size and morphology analysis revealed the formation of ~ 8.4 ± 0.2-nm NMC as measured by dynamic light scattering. We compared the antiviral activity of NMC with molecular monocaprin (MMC) at 0.5 mM and 2 mM concentrations against phi6, which we used as a surrogate for SARS-CoV-2. The synthesized NMC exhibited 50% higher antiviral activity against phi6 than MMC at pH 7 using plaque assay. NMC inactivated phi6 stronger at pH 4 than at pH 7. To determine if NMC is toxic to mammalian cells, we used MTS assay to assess its IC50 for HPDE and HeLa cell lines, which were ~ 203 and 221 µM, respectively. NMC may be used for prophylactic application either as a drop or spray since many viruses enter the human body through the mucosal lining of the nose, eyes, and lungs.

Influence of silver ion release on the inactivation of antibiotic resistant bacteria using light-activated silver nanoparticles.

The widespread increase in antibiotic resistance (AR), in an extensive range of microorganisms, demands the development of alternative antimicrobials with novel non-specific low-mutation bacterial targets. Silver nanoparticles (AgNPs) and photosensitizers (PSs) are promising antimicrobial agents with broad-spectrum activity and low tendency for antimicrobial resistance development. Herein, we investigated the light-mediated oxidation of AgNPs for accelerated release of Ag+ in the antibacterial synergy of PS-AgNP conjugates using protoporphyrin IX (PpIX) as a PS. Also, the influence of polyethyleneimine (PEI) coated AgNPs in promoting antibacterial activity was examined. We synthesized, characterized and tested the antimicrobial effect of three nanoparticles: AgNPs, PpIX-AgNPs, and PEI-PpIX-AgNPs against a methicillin-resistant Staphylococcus aureus strain (MRSA) and a wild-type multidrug resistant (MDR) E. coli. PpIX-AgNPs were the most effective material achieving >7 log inactivation of MRSA and MDR E. coli. The order of bacterial log inactivation was PpIX-AgNPs > PEI-PpIX-AgNPs > AgNPs. This order correlates with the trend of Ag+ concentration released by the NPs (PpIX-AgNPs > PEI-PpIX-AgNPs > AgNPs). Our study confirms a synergistic effect between PpIX and AgNPs in the inactivation of AR pathogens with about 10-fold increase in inactivation of ARB relative to AgNPs only. The concentration of Ag+ released from NPs determined the log inactivation of MRSA and MDR E. coli more than either the phototoxic effect or the electrostatic interaction promoted by surface charge of nanoparticles with bacteria cells. All NPs showed negligible cytotoxicity to mammalian cells at the bacterial inhibitory concentration after 24 h exposure. These observations confirm the crucial role of optimized Ag+ release for enhanced performance of AgNP-based antimicrobials against AR pathogens.

Tracking the temporal variation of COVID-19 surges through wastewater-based epidemiology during the peak of the pandemic: A six-month long study in Charlotte, North Carolina.

The global spread of SARS-CoV-2 has continued to be a serious concern after WHO declared the virus to be the causative agent of the coronavirus disease 2019 (COVID-19) a global pandemic. Monitoring of wastewater is a useful tool for assessing community prevalence given that fecal shedding of SARS-CoV-2 occurs in high concentrations by infected individuals, regardless of whether they are asymptomatic or symptomatic. Using tools that are part of wastewater-based epidemiology (WBE) approach, combined with molecular analyses, wastewater monitoring becomes a key piece of information used to assess trends and quantify the scale and dynamics of COVID-19 infection in a specific community, municipality, or area of service. This study investigates a six-month long SARS-CoV-2 RNA quantification in influent wastewater from four municipal wastewater treatment plants (WWTP) serving the Charlotte region of North Carolina (NC) using both RT-qPCR and RT-ddPCR platforms. Influent wastewater was analyzed for the nucleocapsid (N) genes N1 and N2. Both RT-qPCR and RT-ddPCR performed well for detection and quantification of SARS-CoV-2 using the N1 target, while for the N2 target RT-ddPCR was more sensitive. SARS-CoV-2 concentration ranged from 103 to 105 copies/L for all four plants. Both RT-qPCR and RT-ddPCR showed a significant positive correlation between SARS-CoV-2 concentrations and the 7-day rolling average of clinically reported COVID-19 cases when lagging 5 to 12 days (ρ = 0.52-0.92, p < 0.001-0.02). A major finding of this study is that RT-qPCR and RT-ddPCR generated SARS-CoV-2 data that was positively correlated (ρ = 0.569, p < 0.0001) and can be successfully used to monitor SARS-CoV-2 signals across the WWTP of different sizes and metropolitan service functions without significant anomalies.

Performance evaluation of virus concentration methods for implementing SARS-CoV-2 wastewater based epidemiology emphasizing quick data turnaround.

Wastewater based epidemiology (WBE) has drawn significant attention as an early warning tool to detect and predict the trajectory of COVID-19 cases in a community, in conjunction with public health data. This means of monitoring for outbreaks has been used at municipal wastewater treatment centers to analyze COVID-19 trends in entire communities, as well as by universities and other community living environments to monitor COVID-19 spread in buildings. Sample concentration is crucial, especially when viral abundance in raw wastewater is below the threshold of detection by RT-qPCR analysis. We evaluated the performance of a rapid ultrafiltration-based virus concentration method using InnovaPrep Concentrating Pipette (CP) Select and compared this to the established electronegative membrane filtration (EMF) method. We evaluated sensitivity of SARS-CoV-2 quantification, surrogate virus recovery rate, and sample processing time. Results suggest that the CP Select concentrator is more efficient at concentrating SARS-CoV-2 from wastewater compared to the EMF method. About 25% of samples that tested negative when concentrated with the EMF method produced a positive signal with the CP Select protocol. Increased recovery of the surrogate virus control using the CP Select confirms this observation. We optimized the CP Select protocol by adding AVL lysis buffer and sonication, to increase the recovery of virus. Sonication increased Bovine Coronavirus (BCoV) recovery by 19%, which seems to compensate for viral loss during centrifugation. Filtration time decreases by approximately 30% when using the CP Select protocol, making this an optimal choice for building surveillance applications where quick turnaround time is necessary.

Nanoantibiotics: Functions and Properties at the Nanoscale to Combat Antibiotic Resistance.

One primary mechanism for bacteria developing resistance is frequent exposure to antibiotics. Nanoantibiotics (nAbts) is one of the strategies being explored to counteract the surge of antibiotic resistant bacteria. nAbts are antibiotic molecules encapsulated with engineered nanoparticles (NPs) or artificially synthesized pure antibiotics with a size range of ≤100 nm in at least one dimension. NPs may restore drug efficacy because of their nanoscale functionalities. As carriers and delivery agents, nAbts can reach target sites inside a bacterium by crossing the cell membrane, interfering with cellular components, and damaging metabolic machinery. Nanoscale systems deliver antibiotics at enormous particle number concentrations. The unique size-, shape-, and composition-related properties of nAbts pose multiple simultaneous assaults on bacteria. Resistance of bacteria toward diverse nanoscale conjugates is considerably slower because NPs generate non-biological adverse effects. NPs physically break down bacteria and interfere with critical molecules used in bacterial processes. Genetic mutations from abiotic assault exerted by nAbts are less probable. This paper discusses how to exploit the fundamental physical and chemical properties of NPs to restore the efficacy of conventional antibiotics. We first described the concept of nAbts and explained their importance. We then summarized the critical physicochemical properties of nAbts that can be utilized in manufacturing and designing various nAbts types. nAbts epitomize a potential Trojan horse strategy to circumvent antibiotic resistance mechanisms. The availability of diverse types and multiple targets of nAbts is increasing due to advances in nanotechnology. Studying nanoscale functions and properties may provide an understanding in preventing future outbreaks caused by antibiotic resistance and in developing successful nAbts.

Implementing building-level SARS-CoV-2 wastewater surveillance on a university campus.

The COVID-19 pandemic has been a source of ongoing challenges and presents an increased risk of illness in group environments, including jails, long-term care facilities, schools, and residential college campuses. Early reports that the SARS-CoV-2 virus was detectable in wastewater in advance of confirmed cases sparked widespread interest in wastewater-based epidemiology (WBE) as a tool for mitigation of COVID-19 outbreaks. One hypothesis was that wastewater surveillance might provide a cost-effective alternative to other more expensive approaches such as pooled and random testing of groups. In this paper, we report the outcomes of a wastewater surveillance pilot program at the University of North Carolina at Charlotte, a large urban university with a substantial population of students living in on-campus dormitories. Surveillance was conducted at the building level on a thrice-weekly schedule throughout the university's fall residential semester. In multiple cases, wastewater surveillance enabled the identification of asymptomatic COVID-19 cases that were not detected by other components of the campus monitoring program, which also included in-house contact tracing, symptomatic testing, scheduled testing of student athletes, and daily symptom reporting. In the context of all cluster events reported to the University community during the fall semester, wastewater-based testing events resulted in the identification of smaller clusters than were reported in other types of cluster events. Wastewater surveillance was able to detect single asymptomatic individuals in dorms with resident populations of 150-200. While the strategy described was developed for COVID-19, it is likely to be applicable to mitigation of future pandemics in universities and other group-living environments.

Antibiotic resistance development and human health risks during wastewater reuse and biosolids application in agriculture.

The reuse of treated wastewater (TWW) and sewage sludge are considered as solutions to the limited water resource and sludge disposal issues, respectively. The associated environmental and human health risks need to be analyzed to assess whether they are safe solutions or not. This paper discusses issues that relate to the accumulation of antibiotics and antibiotic resistance (AR) determinants in agricultural lands and crops, following TWW irrigation and biosolid amendment. Exposure assessment and dose-response assessment are the two important aspects of risk assessment discussed in this paper. Finally, research gaps in current knowledge that are relevant to a comprehensive and quantitative AR risk assessment were identified which includes: 1.) Studies on soil conditions that increase the frequency of horizontal gene transfer (HGT) between native soil resistome and pathogenic microbes in biosolids and TWW 2.) Holistic studies that examine the accumulation or dissipation of antibiotics, antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) from the irrigation/biosolids application stage to crop consumption stage 3.) The influences of soil environmental conditions (e.g. salinity, nutrients) on the fate of ARB and ARGs in soil and translocation in edible plants 4.) The development of dose-response models that explicitly incorporate the potential for ARGs transfer between microbes when quantifying the risks of infection due to ARB.

Characterization of extracellular polymeric substances in biofilms under long-term exposure to ciprofloxacin antibiotic using fluorescence excitation-emission matrix and parallel factor analysis.

The presence of antibiotic residues in the environment has been regarded as an emerging concern due to their potential adverse environmental consequences such as antibiotic resistance. However, the interaction between antibiotics and extracellular polymeric substances (EPSs) of biofilms in wastewater treatment systems is not entirely clear. In this study, the effect of ciprofloxacin (CIP) antibiotic on biofilm EPS matrix was investigated and characterized using fluorescence excitation-emission matrix (EEM) and parallel factor (PARAFAC) analysis. Physicochemical analysis showed that the proteins were the major EPS fraction, and their contents increased gradually with an increase in CIP concentration (0-300 µg/L). Based on the characterization of biofilm tightly bound EPS (TB-EPS) by EEM, three fluorescent components were identified by PARAFAC analysis. Component C1 was associated with protein-like substances, and components C2 and C3 belonged to humic-like substances. Component C1 exhibited an increasing trend as the CIP addition increased. Pearson's correlation results showed that CIP correlated significantly with the protein contents and component C1, while strong correlations were also found among UV254, dissolved organic carbon, humic acids, and component C3. A combined use of EEM-PARAFAC analysis and chemical measurements was demonstrated as a favorable approach for the characterization of variations in biofilm EPS in the presence of CIP antibiotic.

Diversity of DNA viruses in effluents of membrane bioreactors in Traverse City, MI (USA) and La Grande Motte (France).

This study assesses diversity of DNA viruses in the effluents of two membrane bioreactor (MBR) wastewater treatment plants (WWTPs): an MBR in the United States and an MBR in France. Viral diversity of these effluents is compared to that of a conventional activated sludge WWTP in the U.S. Diversity analysis indicates Herpesvirales to be the most abundant order of potentially pathogenic human DNA viruses in wastewater treated effluent in all utilities. Other potentially pathogenic human viruses detected include Adenoviridae, Parvoviridae, and Polyomaviridae. Bacteriophage order Caudovirales comprises the majority of DNA virus sequences in the effluent of all utilities. The choice of treatment process (MBR versus activated sludge reactor) utilized had no impact on effluent DNA viral diversity. In contrast, the type of disinfection applied had an impact on the viral diversity present in the effluent.

Strategies to Combat Antibiotic Resistance in the Wastewater Treatment Plants.

The main goal of this manuscript is to review different treatment strategies and mechanisms for combating the antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARGs) in the wastewater environment. The high amount of antibiotics is released into the wastewater that may promote selection of ARB and ARGs which find their way into natural environments. Emerging microbial pathogens and increasing antibiotic resistance among them is a global public health issue. The propagation and spread of ARB and ARGs in the environment may result in an increase of antibiotic resistant microbial pathogens which is a worldwide environmental and public health concern. A proper treatment of wastewater is essential before its discharge into rivers, lake, or sewage system to prevent the spread of ARB and ARGs into the environment. This review discusses various treatment options applied for combating the spread of ARB and ARGs in wastewater treatment plants (WWTPs). It was reported that low-energy anaerobic-aerobic treatment reactors, constructed wetlands, and disinfection processes have shown good removal efficiencies. Nanomaterials and biochar combined with other treatment methods and coagulation process are very recent strategies regarding ARB and ARGs removal and need more investigation and research. Based on current studies a wide-ranging removal efficiency of ARGs can be achieved depending on the type of genes present and treatment processes used, still, there are gaps that need to be further investigated. In order to find solutions to control dissemination of antibiotic resistance in the environment, it is important to (1) study innovative strategies in large scale and over a long time to reach an actual evaluation, (2) develop risk assessment studies to precisely understand occurrence and abundance of ARB/ARGs so that their potential risks to human health can be determined, and (3) consider operating and environmental factors that affect the efficiency of each treatment mechanism.

Correlation of tetracycline and sulfonamide antibiotics with corresponding resistance genes and resistant bacteria in a conventional municipal wastewater treatment plant.

Antibiotics and corresponding resistance genes and resistant bacteria have been considered as emerging pollutants worldwide. Wastewater treatment plants (WWTPs) are potential reservoirs contributing to the evolution and spread of antibiotic resistance. In this study, total concentrations of tetracycline and sulfonamide antibiotics in final effluent were detected at 652.6 and 261.1ng/L, respectively, and in treated sludge, concentrations were at 1150.0 and 76.0µg/kg dry weight (dw), respectively. The quantities of antibiotic resistance genes and antibiotic resistant bacteria in final effluent were quantified in the range of 9.12×10(5)-1.05×10(6) gene abundances /100mL (genomic copies/100mL) and 1.05×10(1)-3.09×10(3)CFU/mL, respectively. In treated sludge, they were quantified at concentrations of 1.00×10(8)-1.78×10(9) gene abandances/100mL and 7.08×10(6)-1.91×10(8)CFU/100mL, respectively. Significant reductions (2-3 logs, p<0.05) of antibiotic resistance genes and antibiotic resistant bacteria were observed between raw influent and final effluent. The gene abundances of tetO and tetW normalized to that of 16S rRNA genes indicated an apparent decrease as compared to sulI genes, which remained stable along each treatment stage. Significant correlations (R(2)=0.75-0.83, p<0.05) between numbers of resistant bacteria and antibiotic concentrations were observed in raw influent and final effluent. No significance (R(2)=0.15, p>0.05) was found between tet genes (tetO and tetW) with concentration of tetracyclines identified in wastewater, while a significant correlation (R(2)=0.97, p<0.05) was observed for sulI gene and total concentration of sulfonamides. Correlations of the quantities of antibiotic resistance genes and antibiotic resistant bacteria with corresponding concentrations of antibiotics in sludge samples were found to be considerably weak (R(2)=0.003-0.07).

Levels of antibiotic resistance genes in manure, biosolids, and fertilized soil.

Increasing antibiotic resistance genes in the environment may pose a threat to public health. In this study, tetracycline and sulfonamide resistance genes (Tet-W, Tet-O, and Sul-I) were quantified in 24 manure samples from three farms and 18 biosolids samples from seven different wastewater treatment plants using quantitative polymerase chain reaction methods. Concentrations of Tet-W and Tet-O genes were observed to be significantly higher (p < 0.05) in manure than in biosolids samples. The background soil samples showed significantly lower concentration of the above genes compared with manure and biosolids. Lime-stabilized biosolids showed significantly (p < 0.05) lower concentration of antibiotic resistance genes compared with other biosolids treatment methods. Elevated levels of antibiotic resistance genes (Tet-W, Tet-O, and Sul-I) were observed in the amended soil samples after the land application of manure or biosolids (Site A) monitored for a period of about 4 mo. However, at another site (Site B), no significant increase (p > 0.05) in concentration of antibiotic resistance genes was observed after biosolids application on soil. Even though the concentration of antibiotic resistance genes in manure was statistically higher than that in biosolids, when they were applied on land, the contribution to the soil depended on the background soil concentration and the soil characteristics. Further study of multiple soil samples in various locations is needed.

Release of antibiotic resistant bacteria and genes in the effluent and biosolids of five wastewater utilities in Michigan.

The purpose of this study was to quantify the occurrence and release of antibiotic resistant genes (ARGs) and antibiotic resistant bacteria (ARB) into the environment through the effluent and biosolids of different wastewater treatment utilities including an MBR (Membrane Biological Reactor) utility, conventional utilities (Activated Sludge, Oxidative Ditch and Rotatory Biological Contactors-RBCs) and multiple sludge treatment processes (Dewatering, Gravity Thickening, Anaerobic Digestion and Lime Stabilization). Samples of raw wastewater, pre- and post-disinfected effluents, and biosolids were monitored for tetracycline resistant genes (tetW and tetO) and sulfonamide resistant gene (Sul-I) and tetracycline and sulfonamide resistant bacteria. ARGs and ARB concentrations in the final effluent were found to be in the range of ND(non-detectable)-2.33 × 10(6) copies/100 mL and 5.00 × 10(2)-6.10 × 10(5) CFU/100 mL respectively. Concentrations of ARGs (tetW and tetO) and 16s rRNA gene in the MBR effluent were observed to be 1-3 log less, compared to conventional treatment utilities. Significantly higher removals of ARGs and ARB were observed in the MBR facility (range of removal: 2.57-7.06 logs) compared to that in conventional treatment plants (range of removal: 2.37-4.56 logs) (p < 0.05). Disinfection (Chlorination and UV) processes did not contribute in significant reduction of ARGs and ARB (p > 0.05). In biosolids, ARGs and ARB concentrations were found to be in the range of 5.61 × 10(6)-4.32 × 10(9) copies/g and 3.17 × 10(4)-1.85 × 10(9) CFU/g, respectively. Significant differences (p < 0.05) were observed in concentrations of ARGs (except tetW) and ARB between the advanced biosolid treatment methods (i.e., anaerobic digestion and lime stabilization) and the conventional dewatering and gravity thickening methods.