Wastewater based surveillance system to detect SARS-CoV-2 genetic material for countries with on-site sanitation facilities: an experience from Bangladesh

The presence of SARS-CoV-2 genetic materials in wastewater has become a matter of grave for many countries of the world. Wastewater based epidemiology, in this context, emerged as an important tool in developed countries where proper sewage system is available. Due to the recent shift in the spread of the infection from urban to rural areas, it is now equally important to develop a similar mechanism for rural areas as well. Considering the urgency of the issue a study was conducted in 14 districts of Bangladesh and a total of 238 sewage samples were collected in two different periods from December 2020 to January 2021. We are the first to propose a surveillance system for both urban and rural areas where a proper sewage system is absent. Based on RT-PCR analysis of the water samples, in more than 92% of cases, we found the presence of the SARS-COV-2 gene (ORF1ab, N, and Internal Control-IC). The trend of Ct value varies for different study locations. The spread of genetic material for on-site (m = - 0.0386) sanitation system was found more prominent than that of off-site sewage system (m = 0.0105); which indicated the shift of genetic material from urban to rural areas. Wastewater samples were also measured for physicochemical parameters, including pH (6.30 - 12.50) and temperature (22.10 - 32.60)C. The highest viral titer of 1975 copy/mL in sewage sample was observed in a sample collected from the isolation ward of the SARS-COV-2 hospital. Additionally, a correlation was found between bacterial load and SARS-CoV-2 genetic materials. The results indicated the association of increased Ct values with decreasing number of patients and vice versa. The findings reported in this paper contributed to the field of wastewater-based epidemiology dealing with SARS-COV-2 surveillance for developing countries where proper sewage system is absent and highlighting some of the challenges associated with this approach in such settings. Keywords: On-site Sanitation, Off-site Sanitation, SARS-CoV-2, Genetic Materials, Developing Countries, Rural Wastewater-Based Surveillance System.

A rapid and simple protocol for concentration of SARS-CoV-2 from sewage

The aim of this study was to set up a simple protocol to concentrate SARS-CoV-2 from sewage, which can be implemented in laboratories with minimal equipment resources. The method avoids the need for extensive purification steps and reduces the concentration of potential inhibitors of RT-qPCR contained in sewage. The concentration method consists of a single step, in which a small volume of sewage sample is incubated with polyaluminum chloride (PAC). Virus particles adsorbed to the precipitate are collected by low-speed centrifugation, after which the recovered pellet is resuspended with a saline buffer. The PAC concentration method produced an average shift of 4.4-units in Cq values compared to non-concentrated samples, indicating a 25-fold increase in detection sensitivity. The lower detection limit corresponded approximately to 100 copies per ml. Kappa index indicated substantial agreement between PAC and PEG precipitation protocols (k=0.688, CI 0.457-0.919). PAC concentrated samples can be processed immediately for RNA purification and qPCR or sent refrigerated to a diagnosis center, where SARS-CoV-2 detection should be performed in the same way as for clinical samples. This low cost protocol could be useful to aid in the monitoring of community circulation of SARS-CoV-2, especially in low- and middle-income countries, which do not have massive access to support from specialized labs for sewage surveillance.

Remediation of textile industry organic dye waste by photocatalysis using eggshell impregnated ZnO/CuO nanocomposite

Heterogeneous photocatalysis using nanocomposites is of great research interest in the treatment of industrial wastewater. The impregnated photocatalyst was produced by liquid state reaction of ZnO/CuO nanocomposite with extracted eggshells. The structure, functional group, metal composition, bandgap, and photocatalytic activity of the nanocomposites were characterized by using XRD, FTIR, AAS, and UV-vis spectroscopy, respectively in the absence and presence of eggshells. Photocatalytic degradation activities of the nanocomposites under UV light irradiation have been tested for a real sewage sample taken from Debre Berhan Textile Industry. From the results, the optimized degradation efficiency of the dye was 97.95% with 0.4 g dose of the photocatalyst, 120 min irradiation time, 120 °C temperature, and pH of 6.7. The results revealed that eggshell impregnated nanocomposite had better catalytic activity than the naked nanocomposite. This is due to the highly porous structure of eggshell biomasses and their sorption characteristics In conclusion when nanocomposites are supported by eggshell biomasses, they are excellent photocatalysts and consecutively it minimizes the contamination of organic dyes from textile effluents.

Screening of Bacteriophage Encoded Toxic Proteins with a Next Generation Sequencing-Based Assay

Bacteriophage vB_EcoM_fHy-Eco03 (fHy-Eco03 for short) was isolated from a sewage sample based on its ability to infect an Escherichia coli clinical blood culture isolate. Altogether, 32 genes encoding hypothetical proteins of unknown function (HPUFs) were identified from the genomic sequence of fHy-Eco03. The HPUFs were screened for toxic properties (toxHPUFs) with a novel, Next Generation Sequencing (NGS)-based approach. This approach identifies toxHPUF-encoding genes through comparison of gene-specific read coverages in DNA from pooled ligation mixtures before electroporation and pooled transformants after electroporation. The performance and reliability of the NGS screening assay was compared with a plating efficiency-based method, and both methods identified the fHy-Eco03 gene g05 product as toxic. While the outcomes of the two screenings were highly similar, the NGS screening assay outperformed the plating efficiency assay in both reliability and efficiency. The NGS screening assay can be used as a high throughput method in the search for new phage-inspired antimicrobial molecules.

Microscopic Observation of SARS-Like Particles in RT-qPCR SARS-CoV-2 Positive Sewage Samples

The ongoing outbreak of novel coronavirus pneumonia (COVID-19) caused by SARS-CoV-2 infection has spread rapidly worldwide. The major transmission routes of SARS-CoV-2 are recognised as inhalation of aerosol/droplets and person-to-person contact. However, some studies have demonstrated that live SARS-CoV-2 can be isolated from the faeces and urine of infected patients, which can then enter the wastewater system. The currently available evidence indicates that the viral RNA present in wastewater may become a potential source of epidemiological data. However, to investigate whether wastewater may present a risk to humans such as sewage workers, we investigated whether intact particles of SARS-CoV-2 were observable and whether it was possible to isolate the virus in wastewater. Using a correlative strategy of light microscopy and electron microscopy (CLEM), we demonstrated the presence of intact and degraded SARS-like particles in RT-qPCR SARS-CoV-2-positive sewage sample collected in the city of Marseille. However, the viral infectivity assessment of SARS-CoV-2 in the wastewater was inconclusive, due to the presence of other viruses known to be highly resistant in the environment such as enteroviruses, rhinoviruses, and adenoviruses. Although the survival and the infectious risk of SARS-CoV-2 in wastewater cannot be excluded from our study, additional work may be required to investigate the stability, viability, fate, and decay mechanisms of SARS-CoV-2 thoroughly in wastewater.

A Modified High-Throughput Screening Protocol to Isolate Bacteriophages from Environmental Samples

In the post antimicrobial era, increasing attention is paid towards using bacteriophage (phage in short) therapy to control antibiotic-resistant bacteria. The first step in phage therapy applications is isolating highly efficient lytic phages or phage cocktails from various sources. When a double-layer- agar with around 0.7% agar in top agar is employed, it results in a low number of phage isolation with a poor resolution, and in many cases, you miss the phage. To address this problem, a low concentration of agar in top agar is examined for better phage isolation. Here, our results proved the efficiency of isolating phage upon formulating a double-layer agar with 0.3% agar in top agar. A sewage sample was collected then phages were isolated, purified, and spotted on a top layer agar with 0.3% agar. The results showed the possibility of isolating a higher number of phages on 0.3% top agar than 0.7%. The finding advocates using 0.3% top agar for the double-layer agar, as it will provide fast, better, and easy phage screening and isolation.

A Modified High-Throughput Screening Protocol to Isolate Bacteriophages from Environmental Samples

In the post antimicrobial era, increasing attention is paid towards using bacteriophage (phage in short) therapy to control antibiotic-resistant bacteria. The first step in phage therapy applications is isolating highly efficient lytic phages or phage cocktails from various sources. When a double-layer- agar with around 0.7% agar in top agar is employed, it results in a low number of phage isolation with a poor resolution, and in many cases, you miss the phage. To address this problem, a low concentration of agar in top agar is examined for better phage isolation. Here, our results proved the efficiency of isolating phage upon formulating a double-layer agar with 0.3% agar in top agar. A sewage sample was collected then phages were isolated, purified, and spotted on a top layer agar with 0.3% agar. The results showed the possibility of isolating a higher number of phages on 0.3% top agar than 0.7%. The finding advocates using 0.3% top agar for the double-layer agar, as it will provide fast, better, and easy phage screening and isolation.

Characterization of a Novel Bacteriophage Henu2 and Evaluation of the Synergistic Antibacterial Activity of Phage-Antibiotics

Staphylococcus aureus phage Henu2 was isolated from a sewage sample collected in Kaifeng, China, in 2017. In this study, Henu2, a linear double-stranded DNA virus, was sequenced and found to be 43,513 bp long with 35% G + C content and 63 putative open reading frames (ORFs). Phage Henu2 belongs to the family Siphoviridae and possesses an isometric head (63 nm in diameter). The latent time and burst size of Henu2 were approximately 20 min and 7.8 plaque forming unit (PFU)/infected cells. The Henu2 maintained infectivity over a wide range of temperature (10–60 °C) and pH values (4–12). Phylogenetic and comparative genomic analyses indicate that Staphylococcus aureus phage Henu2 should be a new member of the family of Siphoviridae class-II. In this paper, Phage Henu2 alone exhibited weak inhibitory activity on the growth of S. aureus. However, the combination of phage Henu2 and some antibiotics or oxides could effectively inhibit the growth of S. aureus, with a decrease of more than three logs within 24 h in vitro. These results provide useful information that phage Henu2 can be combined with antibiotics to increase the production of phage Henu2 and thus enhance the efficacy of bacterial killing.

Time-evolution of SARS-CoV-2 in wastewater during the first pandemic wave of COVID-19 in the metropolitan area of Barcelona.

Two large wastewater treatment plants (WWTP), covering around 2.7 M inhabitants, which represents around 85% of the metropolitan area of Barcelona, were sampled before, during and after the implementation of a complete lockdown. Five one-step RT-qPCR assays, targeting the polymerase (IP2 and IP4), the envelope E and the nucleoprotein (N1 and N2) genome regions, were employed for SARS-CoV-2 RNA detection in 24-h composite wastewater samples concentrated by polyethylene glycol (PEG) precipitation. SARS-CoV-2 was detected in a sewage sample collected 41 days ahead of the declaration of the first COVID-19 case. The evolution of SARS-CoV-2 genome copies in wastewater evidenced the validity of water-based epidemiology to anticipate COVID-19 outbreaks, to evaluate the impact of control measures and even to estimate the burden of shedders, including presymptomatic, asymptomatic, symptomatic and undiagnosed cases. For this latter objective, a model was applied for the estimation of the total number of shedders, evidencing a high proportion of asymptomatic infected individuals. In this way, an infection prevalence of 2.0-6.5% was figured. On the other hand, a proportion of around 0.12% and 0.09% of the total population was determined to be required for positive detection in the two WWTPs. At the end of the lockdown, SARS-CoV-2 RNA apparently disappeared in the WWTPs but could still be detected in grab samples from four urban sewers. Sewer monitoring allowed for location of specific hot spots of COVID-19, enabling the rapid adoption of appropriate mitigation measures. IMPORTANCE Water-based epidemiology (WBE) is a valuable early warning tool for tracking the circulation of the virus among the population, including not only symptomatic patients, but also asymptomatic, presymptomatic and misdiagnosed carriers, which represent a high proportion of the infected population. In the specific case of Barcelona, wastewater surveillance anticipated several weeks not only the original COVID-19 pandemic wave, but also the onset of the second wave. In addition, SARS-CoV-2 occurrence in wastewater evidenced the efficacy of the adopted lockdown measures on the circulation of the virus.