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Since the COVID-19 pandemic started, there has been much speculation about how COVID-19 and antimicrobial resistance may be interconnected. In this study, untreated wastewater was sampled from Hospital A designated to treat COVID-19 patients during the first wave of the COVID-19 pandemic alongside Hospital B that did not receive any COVID-19 patients. Metagenomics was used to determine the relative abundance and mobile potential of antibiotic resistant genes (ARGs), prior to determining the correlation of ARGs with time/incidence of COVID-19. Our findings showed that ARGs resistant to macrolides, sulfonamides, and tetracyclines were positively correlated with time in Hospital A but not in Hospital B. Likewise, minor extended spectrum beta-lactamases (ESBLs) and carbapenemases of classes B and D were positively correlated with time, suggesting the selection of rare and/or carbapenem-resistant genes in Hospital A. Non-carbapenemase

Recent applications of wastewater-based epidemiology (WBE) have demonstrated its ability to track the spread and dynamics of COVID-19 at the community level.

BackgroundRecent applications of wastewater-based epidemiology (WBE) have demonstrated its ability to track the spread and dynamics of COVID-19 at the community level. Despite the growing body of research, quantitative synthesis of SARS-CoV-2 titers in wastewater generated from studies across space and time using diverse methods has not been performed.ObjectiveThe objective of this study is to examine the correlations between SARS-CoV-2 viral titers in wastewater across studies, stratified by key covariates in study methodologies. In addition, we examined the associations of proportions of positive detections (PPD) in wastewater samples and methodological covariates.MethodsWe systematically searched the Web of Science for studies published by February 16th, 2021, performed a reproducible screen, and employed mixed-effects models to estimate the levels of SARS-CoV-2 viral titers in wastewater samples and their correlations to case prevalence, sampling mode (grab or composite sampling), and the fraction of analysis (FOA, i.e., solids, solid-supernatant mixtures, or supernatants/filtrates)ResultsA hundred and one studies were found; twenty studies (1,877 observations) were retained following a reproducible screen. The mean of PPD across all studies was 0.67 (95%-CI, [0.56, 0.79]). The mean titer was 5,244.37 copies/mL (95%-CI, [0; 16,432.65]). The Pearson Correlation coefficients (PCC) between viral titers and case prevalences were 0.28 (95%-CI, [0.01; 0.51) for daily new cases or 0.29 (95%-CI, [-0.15; 0.73]) for cumulative cases. FOA accounted for 12.4% of the variability in PPD, followed by case prevalence (9.3% by daily new cases and 5.9% by cumulative cases) and sampling mode (0.6%). Among observations with positive detections, FOA accounted for 56.0% of the variability in titers, followed by sampling mode (6.9%) and case prevalence (0.9% by daily new cases and 0.8% by cumulative cases). While sampling mode and FOA both significantly correlated with SARS-CoV-2 titers, the magnitudes of increase in PPD associated with FOA were larger. Mixed-effects model treating studies as random effects and case prevalence as fixed effects accounted for over 90% of the variability in SARS-CoV-2 PPD and titers.InterpretationsPositive pooled means and confidence intervals in PCC between SARS-CoV-2 titers and case prevalence indicators provide quantitative evidence reinforcing the value of wastewater-based monitoring of COVID-19. Large heterogeneities among studies in proportions of positive detections, titers, and PCC suggest a strong demand in methods to generate data accounting for cross-study heterogeneities and more detailed metadata reporting. Large variance explained by FOA suggesting FOA as a direction that needs to be prioritized in method standardization. Mixed-effects models accounting for study level variations provide a new perspective to synthesize data from multiple studies.

Extracellular DNA (eDNA) cannot be effectively removed by most of the existing wastewater treatment technologies and can contribute to the gain of new functional traits when transformed into compet...

There is increasing interest in wastewater-based epidemiology (WBE) of SARS-CoV-2 RNA to serve as an early warning system for a community. Despite successful detection of SARS-CoV-2 RNA in wastewaters sampled from multiple locations, there is still no clear idea on the minimal number of cases in a community that are associated with a positive detection of the virus in wastewater. To address this knowledge gap, we sampled wastewaters from a septic tank (n = 57) and biological activated sludge tank (n = 52) located on-site of a hospital. The hospital is providing treatment for SARS-CoV-2 infected patients, with the number of hospitalized patients per day known. It was observed that depending on which nucleocapsid gene is targeted by means of RT-qPCR, a range of 253–409 positive cases out of 10,000 persons are required prior to detecting RNA SARS-CoV-2 in wastewater. There was a weak correlation between N1 and N2 gene abundances in wastewater with the number of hospitalized cases. This correlation was however not observed for N3 gene. The frequency of detecting N1 and N2 gene in wastewater was also higher than that for N3 gene. Furthermore, nucleocapsid genes of SARS-CoV-2 were detected at lower frequency in the partially treated wastewater than in the septic tank. In particular, N1 gene abundance was associated with water quality parameters such as total organic carbon and pH. In instances of positive detection, the average abundance of N1 and N3 genes in the activated sludge tank were reduced by 50 and 70% of the levels detected in septic tank, suggesting degradation of the SARS-CoV-2 gene fragments already occurring in the early stages of the wastewater treatment process., Graphical abstract Image 1, Highlights • 253 to 409 positive cases out of 10,000 persons are required prior to detecting SARS-CoV-2 RNA in wastewater. • N1 and N2 gene abundance exhibit weak positive correlation with number of patients. • N1 gene provides better sensitivity and correlation for use as monitoring marker. • Abundance of N1 gene associated with total organic carbon and pH. • N1 and N3 genes were reduced by > 50% in partially treated wastewater but N2 genes abundance increased.

There is increasing interest to use wastewater-based surveillance of SARS-CoV-2 as an early warning of the outbreak within a community. Despite successful detection of SARS-CoV-2 in wastewaters sampled from multiple locations, there is still no clear idea on the minimal number of cases needed in a community to result in a positive detection of the virus in wastewaters. To address this knowledge gap, we sampled wastewaters from a septic tank and biological activated sludge tank located on-site of a hospital. The hospital is providing treatment for SARS-CoV-2 infected patients, with the number of hospitalized patients per day known. It was observed that > 253 positive cases out of 10,000 persons are required prior to detecting SARS-CoV-2 in wastewater. There was a weak correlation between N1 and N2 gene abundances in wastewater with the number of hospitalized cases. This correlation was however not observed for N3 gene. The occurrence frequency of SARS-CoV-2 is at least 5 times lower in the partially treated wastewater than in the septic tank. Furthermore, abundance of N1 and N3 genes in the activated sludge tank were 50 and 70% of the levels detected in septic tank, suggesting poor persistence of the SARS-CoV-2 gene fragments in wastewater.

Many biological contaminants are disseminated through water, and their occurrence has potential detrimental impacts on public and environmental health. Conventional monitoring tools rely on cultivation and are not robust in addressing modern water quality concerns. This review proposes metagenomics as a means to provide a rapid, nontargeted assessment of biological contaminants in water. When further coupled with appropriate methods (e.g., quantitative PCR and flow cytometry) and bioinformatic tools, metagenomics can provide information concerning both the abundance and diversity of biological contaminants in reclaimed waters., Many biological contaminants are disseminated through water, and their occurrence has potential detrimental impacts on public and environmental health. Conventional monitoring tools rely on cultivation and are not robust in addressing modern water quality concerns. This review proposes metagenomics as a means to provide a rapid, nontargeted assessment of biological contaminants in water. When further coupled with appropriate methods (e.g., quantitative PCR and flow cytometry) and bioinformatic tools, metagenomics can provide information concerning both the abundance and diversity of biological contaminants in reclaimed waters. Further correlation between the metagenomic-derived data of selected contaminants and the measurable parameters of water quality can also aid in devising strategies to alleviate undesirable water quality. Here, we review metagenomic approaches (i.e., both sequencing platforms and bioinformatic tools) and studies that demonstrated their use for reclaimed-water quality monitoring. We also provide recommendations on areas of improvement that will allow metagenomics to significantly impact how the water industry performs reclaimed-water quality monitoring in the future.

The global need for food is posing a serious threat to water security. Treated wastewater can be used as an alternative water supply to mitigate our reliance on nonrenewable waters (defined as water that cannot be replenished within our life span). However, concerns related to emerging contaminants such as antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) can impede efforts to push for widespread use of treated wastewater in agricultural irrigation. This chapter aims to provide a better understanding of the potential concerns by first using case studies in two countries that have already practiced water reuse. Second, we collate and analyze data that suggests that wastewater treatment plants able to achieve at least 8-log reduction in microbiological contaminants may suffice as appropriate intervention barriers for ARB dissemination to the environment. This chapter also recognizes that extracellular DNA-carrying ARGs may not be effectively removed even with membrane-based treatment. There is therefore a need to assess whether extracellular DNA may accumulate in agricultural soils due to repeated use of treated wastewater and to determine the concentrations of extracellular DNA needed to significantly increase horizontal gene transfer (HGT) in the natural environment. Given the large knowledge gaps that hinder an accurate assessment of the associated risks, it would be prudent to adopt the precautionary principle and to implement appropriate intervention strategies and best management practices that minimize the impacts and concerns arising from the reuse of treated wastewater in agriculture.

Extracellular DNA (eDNA) cannot be effectively removed by most of the existing wastewater treatment technologies and can contribute to the gain of new functional traits when transformed into competent bacteria present in downstream environments. This study evaluates the contributions of solar and UV-C

The process of natural transformation allows for the stable uptake, integration, and functional expression of extracellular DNA. This mechanism of horizontal gene transfer has been widely linked to the acquisition of antibiotic resistance and virulence factors. Here, we demonstrate that bromoacetic acid (BAA)-a regulated drinking water disinfection byproduct (DBP)-can stimulate natural transformation rates in the model organism Acinetobacter baylyi ADP1. We demonstrate that transformation stimulation in response to BAA is concentration-dependent and is linked to the ability of this compound to generate DNA damage via oxidative stress. In presence of BAA, transcription of recA was upregulated 20-40% compared to the nontreated controls, indicating that this component of the DNA damage response could be associated with the increase in transformation. Other genes associated with DNA translocation across the cytoplasmic membrane (i.e., pilX, comA) did not exhibit increased transcription in the presence of BAA, indicating that the enhancement of transformation is not associated with increased translocation rates of environmental DNA. Overall, these results lead us to speculate that elevated recA transcription levels could lead to increased integration rates of foreign DNA within the recipient cell during DNA repair. Lastly, we show that an artificial DBP cocktail simulating the environmental concentrations of five water DBP classes stimulates natural transformation by almost 2-fold. The results of this study suggest that mutagens like DBPs may play an important role in enhancing the fixation rates of extracellular DNA in the environmental metagenome.

A monitoring effort that spanned across 1.5 years was conducted to examine three types of produce-associated microbiota. The average amount of antibiotic-resistant bacteria recovered from lettuce, tomato, and cucumber was 1.02 × 1010, 2.05 × 107, and 4.78 × 109 cells per 50 g of each produce, respectively. A total of 480 bacterial isolates were obtained and identified from their 16S rRNA genes, revealing isolates that were ubiquitously recovered from all three types of produce. However, sporadic presence of Klebsiella pneumoniae and Acinetobacter baumannii was detected on lettuce and cucumbers but not tomatoes. End-point PCR revealed that the K. pneumoniae and A. baumannii isolates were positive for genes encoding extended spectrum beta-lactamase. Whole genome sequencing of two of the K. pneumoniae isolates further suggested the presence of the blaCTX-M-15 gene in a conjugative plasmid, as well as other antibiotic resistance genes and virulence-associated traits in either conjugative plasmids or the chromosomal genome. Quantitative microbial risk assessment indicated varying levels of ingestion risk associated with different types of produce. In particular, the risk arising from ESBL-positive K. pneumoniae in lettuce, but not in cucumbers or tomatoes, was higher than the acceptable annual risk of 10−4. Practical applications Three types of vegetables were sampled and evaluated over 1.5 years to determine differences in their associated bacterial isolates. Particular emphasis was placed on identifying pathogenic strains that were positive for extended spectrum beta-lactamase (ESBL). Quantitative estimates of the microbial risk associated with the ESBL-positive pathogens showed that different produce types may incur varying levels of ingestion risk. Most of the currently reported ESBL-positive bacterial isolates have been identified in nosocomial environments. However, the carriage of such drug-resistant bacteria in vegetables suggests a possible connection between our daily diet and human health.

The presence of emerging biological pollutants in treated wastewater effluents has gained attention due to increased interest in water reuse. To evaluate the effectiveness of the removal of such contaminants by the conventional wastewater treatment process, the fate and decay kinetics of NDM-1-positiveEscherichia colistrain PI7 and its plasmid-encoded antibiotic resistance genes (ARGs) were assessed in microcosms of anaerobic and aerobic sludge. Results showed thatE. coliPI7 decayed at a significantly lower rate under anaerobic conditions. Approximate half-lives were 32.4 ± 1.4 h and 5.9 ± 0.9 h in the anaerobic and aerobic microcosms, respectively. In the aerobic microcosms, after 72 h of operation,E. coliPI7 remained detectable, but no further decay was observed. Instead, 1 in every 10,000E. colicells was identified to be recalcitrant to decay and persist indefinitely in the sludge. ARGs associated with theE. coliPI7 strain were detected to have transferred to other native microorganisms in the sludge or were released to the liquid fraction upon host decay. Extracellular DNA quickly degraded in the liquid fraction of the aerobic sludge. In contrast, no DNA decay was detected in the anaerobic sludge water matrix throughout the 24-h sampling period. This study suggests an increased likelihood of environmental dispersion of ARGs associated with anaerobically treated wastewater effluents and highlights the potential importance of persister cells in the dissemination ofE. coliin the environment during reuse events of treated wastewater.IMPORTANCEThis study examines the decay kinetics of a pathogenic and antibiotic resistant strain ofEscherichia coliin microcosms simulating biological treatment units of aerobic and anaerobic sludge. The results of this study point at a significantly prolonged persistence of theE. coliand the associated antibiotic resistance gene in the anaerobic sludge. However, horizontal transfer of the plasmid encoding the antibiotic resistance gene was detected in the aerobic sludge by a cultivation method. A subpopulation of persisterE. colicells was also detected in the aerobic sludge. The findings of this study suggest potential areas of concern arising from pathogenic and antibiotic-resistantE. coliduring both anaerobic and aerobic sludge treatment processes.

This study examined the decay kinetics and molecular responses of two Escherichia coli strains upon solar irradiation. The first is E. coli PI-7, a virulent and antibiotic-resistant strain that was isolated from wastewater and carries the emerging NDM-1 antibiotic resistance gene. The other strain, E. coli DSM1103, displayed lower virulence and antibiotic resistance than E. coli PI-7. In a buffer solution, E. coli PI-7 displayed a longer lag phase prior to decay and a longer half-life compared with E. coli DSM1103 (6.64 ± 0.63 h and 2.85 ± 0.46 min vs 1.33 ± 0.52 h and 2.04 ± 0.36 min). In wastewater, both E. coli strains decayed slower than they did in buffer. Although solar irradiation remained effective in reducing the numbers of both strains by more than 5-log10 in

The emergence of resistance to last-resort antibiotics is a public health concern of global scale. Besides direct person-to-person propagation, environmental pathways might contribute to the dissemination of antibiotic-resistant bacteria and antibiotic resistance genes (ARGs). Here, we describe the incidence of bla NDM-1 , a gene conferring resistance to carbapenems, in the wastewater of the city of Jeddah, Saudi Arabia, over a 1-year period. bla NDM-1 was detected at concentrations ranging from 10 4 to 10 5 copies/m 3 of untreated wastewater during the entire monitoring period. These results indicate the ubiquity and high incidence of bla NDM-1 in the local wastewater. To track the bacteria carrying bla NDM-1 , we isolated Escherichia coli PI7, a strain of sequence type 101 (ST101), from wastewater around the Hajj event in October 2013. Genome sequencing of this strain revealed an extensive repertoire of ARGs as well as virulence and invasive traits. These traits were further confirmed by antibiotic resistance profiling and in vitro cell internalization in HeLa cell cultures. Given that this strain remains viable even after a certain duration in the sewerage, and that Jeddah lacks a robust sanitary infrastructure to fully capture all generated sewage, the presence of this bacterium in the untreated wastewater represents a potential hazard to the local public health. To the best of our knowledge, this is the first report of a bla NDM-1 -positive E. coli strain isolated from a nonnosocomial environment in Saudi Arabia and may set a priority concern for the need to establish improved surveillance for carbapenem-resistant E. coli in the country and nearby regions.