Developing wastewater-based surveillance schemes for multiple pathogens: The WastPan project in Finland.

Wastewater comprises multiple pathogens and offers a potential for wastewater-based surveillance (WBS) to track the prevalence of communicable diseases. The Finnish WastPan project aimed to establish wastewater-based pandemic preparedness for multiple pathogens (viruses, bacteria, parasites, fungi), including antimicrobial resistance (AMR). This article outlines WastPan's experiences in this project, including the criteria for target selection, sampling locations, frequency, analysis methods, and results communication. Target selection relied on epidemiological and microbiological evidence and practical feasibility. Within the WastPan framework, wastewater samples were collected between 2021 and 2023 from 10 wastewater treatment plants (WWTPs) covering 40 % of Finland's population. WWTP selection was validated for reported cases of Extended Spectrum Beta-lactamase-producing bacterial pathogens (Escherichia coli and Klebsiella pneumoniae) from the National Infectious Disease Register. The workflow included 24-h composite influent samples, with one fraction for culture-based analysis (bacteria and fungi) and the rest of the sample was reserved for molecular analysis (viruses, bacteria, antibiotic resistance genes, and parasites). The reproducibility of the monitoring workflow was assessed for SARS-CoV-2 through inter-laboratory comparisons using the N2 and N1 assays. Identical protocols were applied to same-day samples, yielding similar positivity trends in the two laboratories, but the N2 assay achieved a significantly higher detection rate (Laboratory 1: 91.5 %; Laboratory 2: 87.4 %) than the N1 assay (76.6 %) monitored only in Laboratory 2 (McNemar, p < 0.001 Lab 1, = 0.006 Lab 2). This result indicates that the selection of monitoring primers and assays may impact monitoring sensitivity in WBS. Overall, the current study recommends that the selection of sampling frequencies and population coverage of the monitoring should be based on pathogen-specific epidemiological characteristics. For example, pathogens that are stable over time may need less frequent annual sampling, while those that are occurring across regions may require reduced sample coverage. Here, WastPan successfully piloted WBS for monitoring multiple pathogens, highlighting the significance of one-litre community composite wastewater samples for assessing community health. The infrastructure established for COVID-19 WBS is valuable for monitoring various pathogens. The prioritization of the monitoring targets optimizes resource utilization. In the future legislative support in target selection, coverage determination, and sustained funding for WBS is recomended.

A comparative analysis employing a gene- and genome-centric metagenomic approach reveals changes in composition, function, and activity in waterworks with different treatment processes and source water in Finland.

The emergence and development of next-generation sequencing technologies (NGS) has made the analysis of the water microbiome in drinking water distribution systems (DWDSs) more accessible and opened new perspectives in microbial ecology studies. The current study focused on the characterization of the water microbiome employing a gene- and genome-centric metagenomic approach to five waterworks in Finland with different raw water sources, treatment methods, and disinfectant. The microbial communities exhibit a distribution pattern of a few dominant taxa and a large representation of low-abundance bacterial species. Changes in the community structure may correspond to the presence or absence and type of disinfectant residual which indicates that these conditions exert selective pressure on the microbial community. The Archaea domain represented a small fraction (up to 2.5%) and seemed to be effectively controlled by the disinfection of water. Their role particularly in non-disinfected DWDS may be more important than previously considered. In general, non-disinfected DWDSs harbor higher microbial richness and maintaining disinfectant residual is significantly important for ensuring low microbial numbers and diversity. Metagenomic binning recovered 139 (138 bacterial and 1 archaeal) metagenome-assembled genomes (MAGs) that had a >50% completeness and <10% contamination consisting of 20 class representatives in 12 phyla. The presence and occurrence of nitrite-oxidizing bacteria (NOB)-like microorganisms have significant implications for nitrogen biotransformation in drinking water systems. The metabolic and functional complexity of the microbiome is evident in DWDSs ecosystems. A comparative analysis found a set of differentially abundant taxonomic groups and functional traits in the active community. The broader set of transcribed genes may indicate an active and diverse community regardless of the treatment methods applied to water. The results indicate a highly dynamic and diverse microbial community and confirm that every DWDS is unique, and the community reflects the selection pressures exerted at the community structure, but also at the levels of functional properties and metabolic potential.

Metagenomic Analysis of the Abundance and Composition of Antibiotic Resistance Genes in Hospital Wastewater in Benin, Burkina Faso, and Finland.

Antibiotic resistance is a global threat to human health, with the most severe effect in low- and middle-income countries. We explored the presence of antibiotic resistance genes (ARGs) in the hospital wastewater (HWW) of nine hospitals in Benin and Burkina Faso, two low-income countries in West Africa, with shotgun metagenomic sequencing. For comparison, we also studied six hospitals in Finland. The highest sum of the relative abundance of ARGs in the 68 HWW samples was detected in Benin and the lowest in Finland. HWW resistomes and mobilomes in Benin and Burkina Faso resembled each other more than those in Finland. Many carbapenemase genes were detected at various abundances, especially in HWW from Burkina Faso and Finland. The blaGES genes, the most widespread carbapenemase gene in the Beninese HWW, were also found in water intended for hand washing and in a puddle at a hospital yard in Benin. mcr genes were detected in the HWW of all three countries, with mcr-5 being the most common mcr gene. These and other mcr genes were observed in very high relative abundances, even in treated wastewater in Burkina Faso and a street gutter in Benin. The results highlight the importance of wastewater treatment, with particular attention to HWW. IMPORTANCE The global emergence and increased spread of antibiotic resistance threaten the effectiveness of antibiotics and, thus, the health of the entire population. Therefore, understanding the resistomes in different geographical locations is crucial in the global fight against the antibiotic resistance crisis. However, this information is scarce in many low- and middle-income countries (LMICs), such as those in West Africa. In this study, we describe the resistomes of hospital wastewater in Benin and Burkina Faso and, as a comparison, Finland. Our results help to understand the hitherto unrevealed resistance in Beninese and Burkinabe hospitals. Furthermore, the results emphasize the importance of wastewater management infrastructure design to minimize exposure events between humans, HWW, and the environment, preventing the circulation of resistant bacteria and ARGs between humans (hospitals and community) and the environment.

Detection and quantification of SARS-CoV-2 RNA in wastewater influent in relation to reported COVID-19 incidence in Finland.

Wastewater-based surveillance is a cost-effective concept for monitoring COVID-19 pandemics at a population level. Here, SARS-CoV-2 RNA was monitored from a total of 693 wastewater (WW) influent samples from 28 wastewater treatment plants (WWTP, N = 21-42 samples per WWTP) in Finland from August 2020 to May 2021, covering WW of ca. 3.3 million inhabitants (∼ 60% of the Finnish population). Quantity of SARS-CoV-2 RNA fragments in 24 h-composite samples was determined by using the ultrafiltration method followed by nucleic acid extraction and CDC N2 RT-qPCR assay. SARS-CoV-2 RNA signals at each WWTP were compared over time to the numbers of confirmed COVID-19 cases (14-day case incidence rate) in the sewer network area. Over the 10-month surveillance period with an extensive total number of samples, the detection rate of SARS-CoV-2 RNA in WW was 79% (including 6% uncertain results, i.e., amplified only in one out of four, two original and two ten-fold diluted replicates), while only 24% of all samples exhibited gene copy numbers above the quantification limit. The range of the SARS-CoV-2 detection rate in WW varied from 33% (including 10% uncertain results) in Pietarsaari to 100% in Espoo. Only six out of 693 WW samples were positive with SARS-COV-2 RNA when the reported COVID-19 case number from the preceding 14 days was zero. Overall, the 14-day COVID-19 incidence was 7.0, 18, and 36 cases per 100 000 persons within the sewer network area when the probability to detect SARS-CoV-2 RNA in wastewater samples was 50%, 75% and 95%, respectively. The quantification of SARS-CoV-2 RNA required significantly more COVID-19 cases: the quantification rate was 50%, 75%, and 95% when the 14-day incidence was 110, 152, and 223 COVID-19 cases, respectively, per 100 000 persons. Multiple linear regression confirmed the relationship between the COVID-19 incidence and the SARS-CoV-2 RNA quantified in WW at 15 out of 28 WWTPs (overall R2 = 0.36, p < 0.001). At four of the 13 WWTPs where a significant relationship was not found, the SARS-CoV-2 RNA remained below the quantification limit during the whole study period. In the five other WWTPs, the sewer coverage was less than 80% of the total population in the area and thus the COVID-19 cases may have been inhabitants from the areas not covered. Based on the results obtained, WW-based surveillance of SARS-CoV-2 could be used as an indicator for local and national COVID-19 incidence trends. Importantly, the determination of SARS-CoV-2 RNA fragments from WW is a powerful and non-invasive public health surveillance measure, independent of possible changes in the clinical testing strategies or in the willingness of individuals to be tested for COVID-19.

Bacterial Genes Encoding Resistance Against Antibiotics and Metals in Well-Maintained Drinking Water Distribution Systems in Finland.

Information on the co-occurrence of antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) among bacterial communities in drinking water distribution systems (DWDSs) is scarce. This study characterized ARGs and MRGs in five well-maintained DWDSs in Finland. The studied DWDSs had different raw water sources and treatment methods. Two of the waterworks employed artificially recharged groundwater (ARGW) and used no disinfection in the treatment process. The other three waterworks (two surface and one groundwater source) used UV light and chlorine during the treatment process. Ten bulk water samples (two from each DWDS) were collected, and environmental DNA was extracted and then sequenced using the Illumina HiSeq platform for high-throughput shotgun metagenome sequencing. A total of 430 ARGs were characterized among all samples with the highest diversity of ARGs identified from samples collected from non-disinfected DWDSs. Furthermore, non-disinfected DWDSs contained the highest diversity of bacterial communities. However, samples from DWDSs using disinfectants contained over double the ratio of ARG reads to 16S rRNA gene reads and most of the MRG (namely mercury and arsenic resistance genes). The total reads and types of ARGs conferring genes associated with antibiotic groups namely multidrug resistance, and bacitracin, beta-lactam, and aminoglycoside and mercury resistance genes increased in waterworks treating surface water with disinfection. The findings of this study contribute toward a comprehensive understanding of ARGs and MRGs in DWDSs. The occurrence of bacteria carrying antibiotic or metal resistance genes in drinking water causes direct exposure to people, and thus, more systematic investigation is needed to decipher the potential effect of these resistomes on human health.