Pathogen Surveillance in Wastewater

A special issue of Microorganisms (ISSN 2076-2607). This special issue belongs to the section "Public Health Microbiology".

Deadline for manuscript submissions: 30 September 2026 | Viewed by 3853

Editors


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Guest Editor
Department of Biochemistry & Microbiology, University of Venda, Thohoyandou, South Africa
Interests: antibiotic

E-Mail Website
Guest Editor
Department of Biochemistry and Microbiology, University of Venda, Thohoyandou, South Africa
Interests: microbiology

Special Issue Information

Dear Colleagues,

The investigation of pathogenic microorganisms present in wastewater and their epidemiological patterns has great importance for public safety, and accordingly, specialized research has been carried out in this area.

The term “microorganism” covers a broad range of living organisms and infectious agents, including bacteria, viruses, protozoa and parasitic worms. Wastewater contains a high concentration of micro-organisms that may be beneficial, neutral or harmful. Fecal contamination of water supplies with pathogenic microorganisms can cause a wide range of human diseases classified as waterborne, foodborne or feco-oral. Several technologies exist to protect the population from exposure to pathogenic microorganisms; however, the implementation of these technologies is often lacking, especially in the developing regions of the world. Collection and treatment of domestic wastewater are essential in such regions to reduce the transmission of infectious diseases. Inadequately treated wastewater when discharged into natural water bodies adversely affects the receiving environment and poses a threat to the biodiversity of aquatic life.

Wastewater-based epidemiology (WBE) has recently emerged as a method to analyze community health by monitoring biological or chemical signatures contained in sewage. WBE provides supplementary information on disease presence and severity trends, monitoring pathogenic microbes in a population reveals concentrations at the source and enables estimation of infection levels after appropriate normalization, particularly during early pandemic stages. Wastewater monitoring thus offers an opportunity to estimate COVID-19 spread within a community and provide an early-warning system for associated disease outbreaks. Additionally, biological toxins and chemicals of interest, including pharmaceuticals, can be monitored concurrently through WBE. Wastewater analysis holds significant potential for epidemiological applications.

This Special Issue will deal with pathogen surveillance in wastewater.

Dr. Afsatou Ndama Traore
Prof. Dr. Natasha Potgieter
Guest Editors

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Keywords

  • microorganisms
  • wastewater
  • public safety

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Published Papers (5 papers)

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Research

22 pages, 1476 KB  
Article
Wastewater Surveillance: A National Concept for Germany—A Refined Approach to Surveillance Site Selection
by Thomas Exner, Ines Flügel, Timo Greiner, Marcus Lukas, Nathan Obermaier, Peter Pütz, Cristina J. Saravia, Alexander Schattschneider, Antje Ullrich and Ulrike Braun
Microorganisms 2026, 14(6), 1197; https://doi.org/10.3390/microorganisms14061197 - 26 May 2026
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Abstract
During the coronavirus disease-2019 (COVID-19) pandemic, wastewater-based surveillance (WBS) gained renewed importance by enabling nationwide assessment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) circulation independent of individual testing. In Germany, WBS was established by a series of initiatives, including the AMELAG project, [...] Read more.
During the coronavirus disease-2019 (COVID-19) pandemic, wastewater-based surveillance (WBS) gained renewed importance by enabling nationwide assessment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) circulation independent of individual testing. In Germany, WBS was established by a series of initiatives, including the AMELAG project, established in 2022. As the pandemic phase waned, the extensive surveillance infrastructure—comprising around 170 wastewater treatment plants (WWTPs)—was scaled down. A subset of 53 WWTPs was selected by a hierarchical set of criteria to ensure continuation of WBS in 2025, resulting in a reduced population coverage (26% instead of 37%), preserving the monitoring of infection dynamics while improving operational efficiency. The multi-stage selection approach integrated operational experience and performance data of WWTPs collected between November 2022 and July 2024, including wastewater characteristics and laboratory quantification success, minimum population coverage across all administrative regional states of the country, and statistical quality metrics such as the frequency of outliers and implausible inflexion points or the deviation from LOESS regression trends. Additional consideration was given to sites of extended research relevance. Reducing the number of WWTPs by more than two-thirds did not result in notable deviations in the aggregated national SARS-CoV-2 viral load profile. However, the evaluation was limited to SARS-CoV-2 data, despite ongoing expansion of the WBS network to include additional pathogens. Overall, the data-driven site-selection framework, developed from scientific and operational criteria, ensures the sustainable continuation of the nationwide WBS system. Full article
(This article belongs to the Special Issue Pathogen Surveillance in Wastewater)
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17 pages, 3089 KB  
Article
Occurrence of Escherichia coli Pathotypes and Antimicrobial Resistance in Wastewater Effluent and Receiving Surface Waters in the Vhembe District, South Africa
by Tshedza Mashamba, Johannes N. T. Mthembu, Vhukhudo Makhomu, Damien Jacobs, Mpumelelo Rikhotso, Leonard Kachienga, Natasha Potgieter and Afsatou N. Traore
Microorganisms 2026, 14(5), 1041; https://doi.org/10.3390/microorganisms14051041 - 4 May 2026
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Abstract
Wastewater treatment plants (WWTPs) are identified as contributors to faecal pollution and the spread of antimicrobial resistance (AMR) in water ecosystems. This research examined the prevalence, profiles of antimicrobial resistance, and pathogenic types of Escherichia coli in effluent from WWTPs and nearby river [...] Read more.
Wastewater treatment plants (WWTPs) are identified as contributors to faecal pollution and the spread of antimicrobial resistance (AMR) in water ecosystems. This research examined the prevalence, profiles of antimicrobial resistance, and pathogenic types of Escherichia coli in effluent from WWTPs and nearby river systems in the Vhembe District. Between May and June 2025, 28 water samples were collected from two WWTP discharge points as well as upstream and downstream locations along the Mvudi, Luvuvhu, and Madadzhe Rivers. The enumeration of E. coli was conducted using Colilert Quanti-Tray method, with isolates obtained via membrane filtration and confirmed using API 20E and VITEK®2 systems. Antimicrobial susceptibility was assessed using VITEK®2, while pathotypes were detected through multiplex PCR. E. coli was found at all sampling locations; however, differences in concentrations across sampling sites and sampling periods were not statistically significant (p > 0.05). Out of 26 confirmed isolates, a significant resistance to β-lactam antibiotics was noted, especially ampicillin (100%). Pathotype analysis revealed strains such as ETEC, EAEC, and EPEC. These results underline extensive contamination by antimicrobial-resistant E. coli in rivers affected by WWTP discharge, which poses potential public health concerns and underscores the necessity for enhanced monitoring efforts. Additional research is needed to validate these findings. Full article
(This article belongs to the Special Issue Pathogen Surveillance in Wastewater)
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15 pages, 2679 KB  
Article
Genomic Epidemiology of Antibiotic-Resistant Bacteria Sampled from Metropolitan Wastewater
by Jakobi T. Deslouches, Nathan J. Raabe, Emma G. Mills, Giuseppe Fleres, Nathan R. Wallace, Mohamed H. Yassin and Daria Van Tyne
Microorganisms 2026, 14(5), 961; https://doi.org/10.3390/microorganisms14050961 - 24 Apr 2026
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Abstract
Wastewater surveillance is an effective approach for monitoring populations of antibiotic-resistant bacteria and tracking the spread of antimicrobial resistance (AMR) across different settings. In this study, hospital and municipal wastewater were collected monthly for 12 months from multiple locations in the greater Pittsburgh [...] Read more.
Wastewater surveillance is an effective approach for monitoring populations of antibiotic-resistant bacteria and tracking the spread of antimicrobial resistance (AMR) across different settings. In this study, hospital and municipal wastewater were collected monthly for 12 months from multiple locations in the greater Pittsburgh area to quantify the presence of antibiotic-resistant bacteria and investigate their genomic diversity. After quantitative culturing on six different selective media types, a total of 150 isolates were speciated by 16S rRNA sequencing, which revealed diverse pathogenic and non-pathogenic taxa, including Klebsiella spp. (n = 28), Pseudomonas spp. (n = 20) and Aeromonas spp. (n = 37). A subset of isolates (n = 46) underwent whole genome sequencing, which identified several antibiotic resistance genes of clinical concern, such as blaKPC (n = 17), blaNDM (n = 6) and blaIMP (n = 6), and revealed genetic similarities between wastewater isolates and clinical isolates collected from infected patients at a Pittsburgh-area medical center. In addition, analysis of plasmids carried by wastewater isolates revealed closely related plasmids present in isolates from different species and sampling locations. Overall, these findings suggest that both hospital and municipal wastewater act as interconnected reservoirs of antimicrobial resistance. Integrating wastewater surveillance with clinical and genomic data could enable the early detection of emerging resistance threats and support proactive infection-control strategies. Full article
(This article belongs to the Special Issue Pathogen Surveillance in Wastewater)
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18 pages, 1584 KB  
Article
Development and Validation of a Quantitative RT-qPCR Panel for the Detection and Monitoring of Polioviruses in Wastewater Samples
by Linnet Immaraj, Judy Y. Qiu, Logan A. Brand, Tiejun Gao, Bonita Lee, Michael Parkins, Casey Hubert, Christine O’Grady and Xiaoli Pang
Microorganisms 2026, 14(3), 709; https://doi.org/10.3390/microorganisms14030709 - 21 Mar 2026
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Abstract
Clusters of acute flaccid paralysis (AFP) caused by oral vaccine-derived poliovirus (VDPV) in 2022 and sporadic outbreaks in New York and Gaza highlight the ongoing risk of polio, alongside the persistent global threat posed by wild-type poliovirus. This study aims to develop and [...] Read more.
Clusters of acute flaccid paralysis (AFP) caused by oral vaccine-derived poliovirus (VDPV) in 2022 and sporadic outbreaks in New York and Gaza highlight the ongoing risk of polio, alongside the persistent global threat posed by wild-type poliovirus. This study aims to develop and validate a quantitative reverse transcription PCR (RT-qPCR) panel that employs different primer–probe sets to simultaneously detect vaccine and wild-type poliovirus (WPV) in wastewater. Using an inactivated poliovirus vaccine (IPV) and engineered DNA fragments (eDNAf), the qPCR master mix (MM) performance, assay specificity, sensitivity (limit of detection, LOD), and recovery from IPV-spiked wastewater were evaluated. Compared with two-step RT-qPCR and qScript MM, one-step RT-qPCR with TaqMan MM improved sensitivity for the following polioviruses (PV): Sabin 1 in IPV and the eDNAf of Sabin 1, 2, and 3; WPV1 and WPV3; and poliovirus type 2 (any serotype 2). The LOD for Sabin 1 in IPV was 2.49 copies/PCR, while LODs for eDNAf of polio targets ranged from 1.06 to 3.12 copies/PCR. Sabin 1 recovery from IPV-spiked wastewater ranged from 10.26% to 57.27%. The RT-qPCR panel for poliovirus exhibited good specificity and sensitivity, with moderate viral recovery, enabling rapid implementation of wastewater monitoring for PV as needed. Full article
(This article belongs to the Special Issue Pathogen Surveillance in Wastewater)
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13 pages, 3265 KB  
Article
Seasonal Variations in the Occurrence of SARS-CoV-2 RNA Recovered from Wastewater Treatment Facilities (WWTFs) Within South Africa
by Kingsley Ehi Ebomah, Luyanda Msolo, Velisa Vuyolwethu Qongwe, Okuhle Mayoyo, Piwe Athi Ntlati, Balisa Ngqwala, Nolonwabo Nontongana, Renee Street, Rabia Johnson and Anthony Ifeanyi Okoh
Microorganisms 2026, 14(2), 495; https://doi.org/10.3390/microorganisms14020495 - 18 Feb 2026
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Abstract
Several researchers have documented the occurrence of the unfamiliar severe acute respiratory syndrome coronavirus 2 ribonucleic acid (also known as SARS-CoV-2 RNA) in various raw wastewater (WW) samples analyzed globally. The efficiency of strategic WW-based epidemiology (WBE) approach as a timely cautioning tool [...] Read more.
Several researchers have documented the occurrence of the unfamiliar severe acute respiratory syndrome coronavirus 2 ribonucleic acid (also known as SARS-CoV-2 RNA) in various raw wastewater (WW) samples analyzed globally. The efficiency of strategic WW-based epidemiology (WBE) approach as a timely cautioning tool for human coronavirus disease-2019 (COVID) and other similar outbreaks is highly promising. This strategy offers a cost-effective, population-wide surveillance tool that can detect rising case trends, from days to weeks before clinical reports, thus enabling proactive public health interventions. This study aimed to detect the occurrence of the viral genome in WW over four seasons, which contributes to the database for multi-plant surveillance research in South Africa. About 480 WW influent samples were amassed from ten sampling points situated in nine wastewater treatment facilities (WWTFs) in Amathole District Municipality (ADM) located in the Province of Eastern Cape (EC), South Africa (SA). The study was carried out for a period of one year. Quantitative real-time polymerase chain reaction (i.e., RT-PCR) was operated to identify the viral genomes in the respective total RNA samples. Of the 480 extracted RNA samples, 210 (44%) were positive with viral genome copies (gc) that ranged from 700 to 40,000 GC/mL. Our results were contrasted with existing COVID-19-positive cases throughout the COVID omicron wave in the ECP. Variations in gc were observed across different seasons, with the highest GC observed in winter. In contrast, there were significant inconsistencies in the existing data of COVID-19 clinical cases, thus indicating no connection between both data. However, with more similar studies, advanced innovative WBE strategies could possibly act as prompt warning tools to signal public health officials about potential future outbreaks. Full article
(This article belongs to the Special Issue Pathogen Surveillance in Wastewater)
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