Not a waste: Wastewater surveillance to enhance public health

Domestic wastewater, when collected and evaluated appropriately, can provide valuable health-related information for a community. As a relatively unbiased and non-invasive approach, wastewater surveillance may complement current practices towards mitigating risks and protecting population health. Spurred by the COVID-19 pandemic, wastewater programs are now widely implemented to monitor viral infection trends in sewersheds and inform public health decision-making. This review summarizes recent developments in wastewater-based epidemiology for detecting and monitoring communicable infectious diseases, dissemination of antimicrobial resistance, and illicit drug consumption. Wastewater surveillance, a quickly advancing Frontier in environmental science, is becoming a new tool to enhance public health, improve disease prevention, and respond to future epidemics and pandemics.

Next-generation sequencing for surveillance of antimicrobial resistance and pathogenicity in municipal wastewater treatment plants.

The World Health Organization (WHO) ranks antimicrobial resistance (AMR) and various pathogens among the top 10 health threats. It is estimated that by 2050, the number of human deaths due to AMR will reach 10 million annually. On the other hand, several infectious outbreaks such as SARS, H1N1 influenza, Ebola, Zika fever, and COVID-19 have severely affected human populations worldwide in the last 20 years. These recent global diseases have generated the need to monitor outbreaks of pathogens and AMR to establish effective public health strategies. This review presents AMR and pathogenicity associated with wastewater treatment plants (WWTP), focusing on Next Generation Sequencing (NGS) monitoring as a complementary system to clinical surveillance. In this regard, WWTP may be monitored at three main points. First, at the inlet (raw wastewater or influent) to identify a broad spectrum of AMR and pathogens contained in the excretions of residents served by sewer networks, with a specific spatio-temporal location. Second, at the effluent, to ensure the elimination of AMR and pathogens in the treated water, considering the rising demand for safe wastewater reuse. Third, in sewage sludge or biosolids, since their beneficial use or final disposal can represent a significant risk to public health. This review is divided into two sections to address the importance and implications of AMR and pathogen surveillance in wastewater and WWTP, based on NGS. The first section presents the fundamentals of surveillance techniques applied in WWTP (metataxonomics, metagenomics, functional metagenomics, metaviromics, and metatranscriptomics). Their scope and limitations are analyzed to show how microbiological and qPCR techniques complement NGS surveillance, overcoming its limitations. The second section discusses the contribution of 36 NGS research papers on WWTP surveillance, highlighting the current situation and perspectives. In both sections, research challenges and opportunities are presented.

Antibiotic Resistance and Its Impact on Disease Management.

Antibiotic resistance has been a challenge to the medical fraternity and has had a massive impact on disease management. The overuse of antibiotics and careless prescription by doctors have been one of the primary reasons for the development of antibiotic resistance among the masses. This article draws attention to the significant reasons causing antibiotic resistance, such as overuse, antibiotic resistance genes, and extensive use of antibiotics in agriculture. It also brings forward the challenges posed by antibiotic resistance in the management of various diseases like tuberculosis, COVID-19, and vancomycin-resistant enterococci infections. The article includes a case study that depicts the threat posed by antibiotic resistance in tuberculosis treatment. This article also shows the effects of antibiotic resistance on COVID-19 patient care and treatment. It further includes methods that can be implemented on international levels as well as individual ground levels to curb antibiotic resistance. One of the methods has a recent finding in which proteins produced in the body are being modified and used in treatments to reduce the use of antibiotics, which ultimately serves the goal of curbing antibiotic resistance by reducing overuse.

Antibiotic resistomes in face-mask biofilm along an urban river: Multiple drivers and co-occurrence with human opportunistic pathogens.

Discarded face masks from the global COVID-19 pandemic have contributed significantly to plastic pollution in surface water, whereas their potential as a reservoir for aquatic pollutants is not well understood. Herein, we conducted a field experiment along a human-impacted urban river, investigating the variations of antibiotic resistance genes (ARGs), pathogens, and water-borne contaminants in commonly-used face masks. Results showed that high-biomass biofilms formed on face masks selectively enriched more ARGs than stone biofilm (0.08-0.22 vs 0.07-0.15 copies/16 S rRNA gene copies) from bulk water, which mainly due to unique microbial communities, enhanced horizontal gene transfer, and selective pressure of accumulated contaminants based on redundancy analysis and variation partitioning analysis. Several human opportunistic pathogens (e.g., Acinetobacter, Escherichia-Shigella, Bacillus, and Klebsiella), which are considered potential ARG carriers, were also greatly concentrated in face-mask biofilms, imposing a potential threat to aquatic ecological environment and human health. Moreover, wastewater treatment plant effluents, as an important source of pollutants to urban rivers, further aggravated the abundances of ARGs and opportunistic pathogens in face-mask biofilms. Our findings demonstrated that discarded face masks provide a hotspot for the proliferation and spread of ARGs and pathogens in urban water, highlighting the urgent requirement for implementing stricter regulations in face mask disposal.

Airborne prokaryotes and toxins

Increasing evidence of possible airborne transmission of many microorganisms causing important human diseases such as the causal agent of the current COVID-19 pandemic has led to renewed interest in the field of aeromicrobiology. In this chapter, we examine the diversity of prokaryotes and toxins found in the atmosphere and their public health importance. This chapter among other important information focuses on occupational exposure to airborne prokaryotes and their toxins, antibiotic-resistant bacteria, and antibiotic resistance genes as emerging airborne pollutants of global public health concern and airborne prokaryotes and toxins in bioterrorism. © 2023 Elsevier Inc. All rights reserved.

Pharmaceutically Active Compounds in Water Bodies—Occurrence, Fate, and Toxicity

The presence of pharmaceutically active compounds (PhACs) in water bodies has been considered an issue of global concern due to their high consumption and release into the environment, especially under pandemic conditions such as current COVID-19 situations. Additionally, the appearance of antibiotic-resistant bacteria (ARBs) and antibiotic resistance genes (ARGs) threatens the effectiveness of the pharmaceuticals developed to treat certain diseases. To address this problem, there have been efforts to develop efficient and cost-effective (waste)water treatment methods or to upgrade the existing facilities to regenerate clean water resources. According to the reports available in the literature, the effectiveness of these methods is highly dependent on the applied technology and the type and concentration of the PhACs. The efficiency of these systems can also determine the environmental and ecotoxicological effects expected from the release of these compounds. This chapter aims to summarize and discuss the available literature on the occurrence, environmental concentrations, fate, and possible effects of typical PhACs when introduced into receiving environments. The existing research gaps have also been discussed, and recommendations have been provided for further studies. © 2023, The Author(s), under exclusive license to Springer Nature Switzerland AG.

The Quinolone Resistance Genes in the Bacteriophage DNA Fractions in the Healthy Calf Stool Samples Via qPCR

The One Health approach shows that people, animals, plants, and environmental factors can affect each other. Phages are one of the mobile genetic elements. Quinolones are a critical group of antibiotics for both human and animal health and monitoring their antimicrobial resistance is very important. The aim of the study is to determine the frequency of the quinolone resistance gene in bacteriophage DNA fractions obtained from healthy calf stool samples. In our study, 50 samples from 6-9 months old calves, which were found to be healthy and not treated with any group of antibiotics in Sanliurfa province, were included. DNA isolation was made from phage lysates of stool samples and specific primers were used qnrA, qnrB and qnrS genes. qPCR was performed on LightCycler480. Despite not receiving any antibiotic treatment, qnrB was the most detected gene among the phage DNA fractions detected in 11 calves. While qnrA, qnrB and qnrS quinolone resistance genes were detected together in one sample, qnrB and qnrS resistance genes were found together in two samples. Our data, obtained from the study in Turkiye to search for antimicrobial resistance genes in phage fractions, showed the importance of the One Health approach and determined that it was highly effective in quinolone resistance gene shedding in healthy calves that had never been treated with antibiotics. It has been concluded that in empirical treatment with quinolone, attention should be paid to all living things and unconscious antibiotic use may cause the spread of resistance genes more than expected.Copyright © 2023, Veteriner Fakultesi Dergisi. All rights reserved.

A holistic review on triclosan and triclocarban exposure: Epidemiological outcomes, antibiotic resistance, and health risk assessment.

Triclosan (TCS) and triclocarban (TCC) are antimicrobials that are widely applied in personal care products, textiles, and plastics. TCS and TCC exposure at low doses may disturb hormone levels and even facilitate bacterial resistance to antibiotics. In the post-coronavirus disease pandemic era, chronic health effects and the spread of antibiotic resistance genes associated with TCS and TCC exposure represent an increasing concern. This study sought to screen and review the exposure levels and sources and changes after the onset of the coronavirus disease (COVID-19) pandemic, potential health outcomes, bacterial resistance and cross-resistance, and health risk assessment tools associated with TCS and TCC exposure. Daily use of antimicrobial products accounts for most observed associations between internal exposure and diseases, while secondary exposure at trace levels mainly lead to the spread of antibiotic resistance genes. The roles of altered gut microbiota in multi-system toxicities warrant further attention. Sublethal dose of TCC selects ARGs without obviously increasing tolerance to TCC. But TCS induce persistent TCS resistance and reversibly select antibiotic resistance, which highlights the benefits of minimizing its use. To derive reference doses (RfDs) for humans, more sensitive endpoints observed in populational studies need to be confirmed using toxicological tests. Additionally, the human equivalent dose is recommended to be incorporated into the health risk assessment to reduce uncertainty of extrapolation.

Exposure to benzalkonium chloride disinfectants promotes antibiotic resistance in sewage sludge microbiomes.

Disinfectants are routinely used in human environments to control and prevent the transmission of microbial disease, and this is particularly true during the current COVID-19 crisis. However, it remains unclear whether the increased disinfectant loadings to wastewater treatment plants facilitate the dissemination of antibiotic resistance genes (ARGs) in sewage sludge microbiomes. Here, we investigated the impacts of benzalkonium chlorides (BACs), widely used disinfectants, on ARGs profiles and microbial community structures in sewage sludge by using high-throughput quantitative PCR and Illumina sequencing. A total of 147 unique ARGs and 39 mobile genetic elements (MGEs) were detected in all sewage sludge samples. Our results show that exposure to BACs disinfectants at environmentally relevant concentrations significantly promotes both the diversity and absolute abundance of ARGs in sludge microbiomes, indicating the co-selection of ARGs by BACs disinfectants. The enrichment of ARGs abundance varied from 2.15-fold to 3.63-fold compared to controls. In addition, BACs exposure significantly alters bacterial and protistan communities, resulting in dysbiosis of the sludge microbiota. The Mantel test and Procrustes analysis confirm that bacterial communities are significantly correlated with ARGs profiles under BACs treatments. The structural equation model explains 83.8 % of the total ARGs variation and further illustrates that the absolute abundance of MGEs exerts greater impacts on the variation of absolute abundance of ARGs than microbial communities under BACs exposure, suggesting BACs may promote antibiotic resistance by enhancing the horizontal gene transfer of ARGs across sludge microbiomes. Collectively, our results provide new insights into the proliferation of antibiotic resistance through disinfectant usage during the pandemic and highlight the necessity to minimize the environmental release of disinfectants into the non-target environment for combating antibiotic resistance.

Optimization of methods for sanitary-microbiological and sanitary-parasitological control of wastewater

Introduction. The main reason for the problems of surface reservoirs in many regions of Russia is the non-compliance with sanitary protection zones and the discharge of insufficiently treated wastewater into reservoirs. SanPiN 1.2.3685-21 introduced a list of the new controlled sanitary-microbiological and parasitological safety indicators for disinfected wastewater and surface water bodies. For some indicators, there are no methods of determination in the current methodological documents. In this work, studies were carried out using various methods, including accelerated ones, which make it possible to determine the normalized indicators in wastewater and surface waters. Goals and objectives – optimization of methods for the study of surface and wastewater in the implementation of sanitary-microbiological and sanitary-parasitological control. Materials and methods. Bacteriological, parasitological indicators, as well as coliphages were determined in samples of surface and wastewater. To detect and identify generalized coliform bacteria, E. coli, enterococci, P. aeruginosa and legionella, in addition to membrane filtration and direct seeding methods, test systems (IDEXX) were used: Colilert-18 and Colilert-24, Pseudolert, Enterolert, Legionelert. Obligate anaerobes of C. perfringens were cultured in an anaerobic box (Anaerobic Station A55, Don Whitley Scientific) in an atmosphere of a three-component gas mixture. Species identification of microorganisms was carried out by time-of-flight mass spectrometry (MALDI TOF MS) using a Microflex mass spectrometer with MALDI BioTyper software (Bruker, Germany). Isolation of RNA/DNA of viruses and bacteria was carried out using a set of reagents “AmpliSens. RIBOT-prep”. Results. As a result of the conducted studies, P. aeruginosa;E. coli, R. ornithinolytica, A. hydrophila, A. caviae, A. molluscorum;E. hirae, E. faecium, E.faecalis;C. perfringens;S. lutetiensis, S. suis were isolated and identified from wastewater at all stages of treatment, high fungal contamination was found, and also Group F adenovirus DNA (both in wastewater and surface water samples) and additionally rotovirus and norovirus RNA, Campylobacter DNA. SarsCov-2 virus RNA was not detected in any sample. Lamblia spp., Blastocystis spp. and Cryptosporidium parvum were identified;opportunistic Entamoeba spp.;helminth eggs – Toxocara spp., Hymenolepis diminuta, Hymenolepis nana, Asagis lumbricoides, egg oncospheres Tenia spp.;Nematodes spp. larvae. Limitations. Since clinically significant strains of bacteria, fungi, and parasitic protozoa were found in the studied water samples, it is important to expand knowledge about water sources as reservoirs and spreaders of these pathogens. Conclusion. Monitoring is now urgently needed to quantify pathogens circulating in surface water and wastewater. © 2022 Izdatel'stvo Meditsina. All rights reserved.

Viral diversity and potential environmental risk in microplastic at watershed scale: Evidence from metagenomic analysis of plastisphere.

Microplastics (MPs) have been considered as a new vector for the long-distance transport of pathogens in aquatic ecosystems. However, the composition of viral communities attached on MPs and their environmental risk are largely unknown. Here, we profiled the viral diversity and potential risk in five different MPs collected from the Beilun River based on metagenomic analysis. Nearly 2863 million raw reads were produced and assembled, and annotation resulted in the identification of 1719 different species of viruses in MPs. Viruses in polypropylene (PP) displayed the highest diversity, with about 250 specific viruses detected. Source tracking of viruses in MPs by the fast expectation-maximization microbial source tracking method (FEAST) demonstrated that viruses in upstream and downstream MPs are two major sources of viruses in estuary. Furthermore, the MP-type-dependent potential environmental risk of viruses was significant based on both antibiotic resistance genes (ARGs) and virulence factors (VFs) detected in viral metagenomes, and PP was confirmed with the highest potential environmental risk. This study reveals the high diversity and potential environmental risk of viruses in different MPs, and provides an important guidance for future environmental monitoring and understanding the potential risks associated with both viral transmission and MPs pollution.

Accumulation of SARS-CoV-2 RNA in Sewer Biofilms

Wastewater-based epidemiology is a useful tool to exist for normalizing wastewater virus concentrations to the flow and number of people in the sewershed, less information is available regarding the potential for in-sewer processes to impact these observations. The aim of this research was to evaluate SARSCoV-2 accumulation in simulated sewer biofilms. An annular biofilm reactor was continuously fed with raw wastewater from a separate sanitary sewer during periods of high and low COVID-19 incidence. Periodic biofilm samples were collected, and SARSCoV-2 and pepper mottle virus gene copies were quantified via RT-qPCR. During the period of low COVID-19 incidence, SARSCoV-2 was below detection or below the quantitation limit in the sewer biofilms. During the period of high COVID-19 incidence, SARS-CoV-2 gene copies increased in the sewer biofilm across the 4-week study period. Accumulation of similar to 700 genome copies/cm(2) was observed in the biofilm and correlated with cumulative cases in the county. During both study periods, the pepper mottle virus plateaued after 1 week. These results provide insight into the potential for SARS-CoV-2 retardation in sewer biofilms, which may be of interest for interpreting wastewater-based epidemiology data, protecting utility workers, and monitoring infections.

Alterations of fecal antibiotic resistome in COVID-19 patients after empirical antibiotic exposure.

As the COVID-19 pandemic spread globally, the consumption of antibiotics increased. However, no studies exist evaluating the effect of antibiotics use on the antibiotic resistance of intestinal flora in COVID-19 patients during the pandemic. To explore this issue, we collected 15 metagenomic data of fecal samples from healthy controls (HCs) with no use history of antibiotics, 23 metagenomic data of fecal samples from COVID-19 patients who received empirical antibiotics [COVID-19 (abx+)], 18 metagenomic data of fecal samples from antibiotics-naïve COVID-19 patients [COVID-19 (abx-)], and six metagenomic data of fecal samples from patients with community-acquired pneumonia [PC (abx+)] from the Sequence Read Archive database. A total of 513 antibiotic-resistant gene (ARG) subtypes of 18 ARG types were found. Antibiotic treatment resulted in a significant increase in the abundance of ARGs in intestinal flora of COVID-19 patients and markedly altered the composition of ARG profiles. Grouped comparisons of pairs of Bray-Curtis dissimilarity values demonstrated that the dissimilarity of the HC versus the COVID-19 (abx+) group was significantly higher than the dissimilarity of the HC versus the COVID-19 (abx-) group. The mexF, mexD, OXA_209, major facilitator superfamily transporter, and EmrB_QacA family major facilitator transporter genes were the discriminative ARG subtypes for the COVID-19 (abx+) group. IS621, qacEdelta, transposase, and ISCR were significantly increased in COVID-19 (abx+) group; they greatly contributed toward explaining variation in the relative abundance of ARG types. Overall, our data provide important insights into the effect of antibiotics use on the antibiotic resistance of COVID-19 patients during the COVID-19 epidemic.

Triclocarban shifted the microbial communities and promoted the spread of antibiotic resistance genes in nitrifying granular sludge system.

Triclocarban (TCC) is in great market demand especially after the outbreak of COVID-19 pandemic, becoming an emerging pollutant. However, the impacts of TCC on the performance of nitrifying granular sludge system and the occurrence of antibiotic resistance genes (ARGs) were still unknown. This work explored the impacts of different concentrations of TCC on nitrifying granular sludge. Results showed that TCC suppressed the activities of ammonia-oxidizing microorganisms and decreased the abundance of Nitrospira. Adsorption was the main way for the removal of TCC and the biodegradation efficiency of TCC increased to 28.00% under 19.70 mg/L TCC addition. TCC enriched the ARGs and promoted the risks of their transferring in microorganisms. Pseudomonas might not only have strong resistance to TCC, but also propagate ARGs. The removal process of TCC and bacterial communities were important factors to promote the spread of ARGs. Thus, the existence of TCC presented a great environmental risk.

Residual chlorine disrupts the microbial communities and spreads antibiotic resistance in freshwater.

Chlorine disinfection is a key global public health strategy for the prevention and control of diseases, such as COVID-19. However, little is known about effects of low levels of residual chlorine on freshwater microbial communities and antibiotic resistomes. Here, we treated freshwater microcosms with continuous low concentrations of chlorine and quantified the effects on aquatic and zebrafish intestinal microbial communities and antibiotic resistomes, using shotgun metagenome and 16S rRNA gene sequencing. Although chlorine rapidly degraded, it altered the aquatic microbial community composition over time and disrupted interactions among microbes, leading to decreases in community complexity and stability. However, community diversity was unaffected. The majority of ecological functions, particularly metabolic capacities, recovered after treatment with chlorine for 14 d, due to microbial community redundancy. There were also increased levels of antibiotic-resistance gene dissemination by horizontal and vertical gene transfer under chlorine treatment. Although the zebrafish intestinal microbial community recovered from temporary dysbiosis, growth and behavior of zebrafish adults were negatively affected by chlorine. Overall, our findings demonstrate the negative effects of residual chlorine on freshwater ecosystems and highlight a possible long-term risk to public health.

River restoration changes distributions of antibiotics, antibiotic resistance genes, and microbial community.

Although river restoration has been increasingly implemented to restore water quality in ecosystems, its effect on the removal of emerging pollutant antibiotics, and their resultant influence on microbial community structure and functions in river water is still unclear. This study investigated the changes of antibiotics, antibiotic resistant genes (ARGs), microbial communities, and their spatial distributions in a megacity river before and after river restoration. Results indicated that although the restoration activities including riverbed dredging, riverbank hardening, sewage and storm water separation and re-pipelining improved water quality such as by decreasing total phosphorus (TP) content from 4.60 ± 6.38 mg/L in 2018 to 0.98 ± 0.44 mg/L in 2020, the antibiotic concentrations in river water increased. Total antibiotic concentrations in the water samples were higher in 2020 (506.89-6952.50 ng/L) than those in 2018 (137.93-1751.51 ng/L), likely caused by increased usage of antibiotics in 2020 for COVID-19 treatment. The spatial distributions of antibiotics were less varied likely as a result of less retardation and fast mixing during antibiotic transport. The result also found that the abundance of Actinobacteria and Proteobacteria, and their correlations with ARGs increased. The spatial distributions of ARGs and microbial communities became less varied in the river water, consistent with the antibiotic variations before and after river restoration. Physicochemical changes such as decreased TP and dissolved organic carbon content may also be a factor. The results indicated that the current river restoration efforts were not effective in removing antibiotics, and implied that further studies are needed to investigate their subsequent transformation and transport, and to assess their risks to the health of ecosystems.

Increased expression of antibiotic-resistance genes in biofilm communities upon exposure to cetyltrimethylammonium bromide (CTAB) and other stress conditions.

Quaternary ammonium compounds (QAC, e.g., cetyltrimethylammonium bromide, (CTAB)) are widely used as surfactants and disinfectants. QAC already are commonly found in wastewaters, and their concentration could increase, since QAC are recommended to inactivate the SARS-CoV-2 (COVID-19) virus. Exposure of bacteria to QAC can lead to proliferation of antibiotic resistance genes (ARG). In particular, O2-based membrane biofilm reactors (O2-MBfRs) achieved excellent CTAB biodegradation, but ARG increased in their biofilms. Here, we applied meta-transcriptomic analyses to assess the impacts of CTAB exposure and operating conditions on microbial community's composition and ARG expression in the O2-MBfRs. Two opportunistic pathogens, Pseudomonas aeruginosa and Stenotrophomonas maltophilia, dominated the microbial communities and were associated with the presence of ARG. Operating conditions that imposed stress on the biofilms, i.e., limited supplies of O2 and nitrogen or a high loading of CTAB, led to large increases in ARG expression, particularly for genes conferring antibiotic-target protection. Important within the efflux pumps was the Resistance-Nodulation-Division (RND) family, which may have been active in exporting CTAB from cells. Oxidative stress appeared to be the key factor that triggered ARG proliferation by selecting intrinsically resistant species and accentuating the expression of ARG. Our findings suggest that means to mitigate the spread of ARG, such as shown here in a O2-based membrane biofilm reactor, need to consider the impacts of stressors, including QAC exposure and stressful operating conditions.

A metagenomic-based method to study hospital air dust resistome.

As a symbol of the defense mechanisms that bacteria have evolved over time, the genes that make bacteria resist antibiotics are overwhelmingly present in the environment. Currently, bacterial antibiotic resistance genes (ARGs) in the air are a serious concern. Previous studies have identified bacterial communities and summarized putative routes of transmissions for some dominant hospital-associated pathogens from hospital indoor samples. However, little is known about the possible indoor air ARG transportation. In this study, we mainly surveyed air-conditioner air dust samples under different airflow conditions and analyzed these samples using a metagenomic-based method. The results show air dust samples exhibited a complex resistome, and the average concentration is 0.00042 copies/16S rRNA gene, which is comparable to some other environments. The hospital air-conditioners can form resistome over time and accumulate pathogens. In addition, our results indicate that the Outpatient hall is one of the main ARG transmission sources, which can distribute ARGs to other departments (explains >80% resistome). We believe that the management should focus on ARG carrier genera such as Staphylococcus, Micrococcus, Streptococcus, and Enterococcus in this hospital and our novel evidence-based network strategy proves that plasmid-mediated ARG transfer can occur frequently. Overall, these results provide insights into the characteristics of air dust resistome and possible route for how ARGs are spread in air.