<i>E. coli</i> as an Indicator of Contamination and Health Risk in Environmental Waters

Good public health depends on regular monitoring of water quality as faecal contamination is a serious problem due to the potential for contracting disease. Bacterial contamination in water is measured using indicator organisms, notably Escherichia coli and Enterococci which are used as primary indicators of contamination in fresh and marine water quality, respectively, rather than the total coliforms present. Although most E. coli and Enterococci strains cause only mild infections, their presence is indicative of the potential presence of other more pathogenic organisms which are a danger to human health. The acceptable levels of indicator organisms are defined in legislation and are set for drinking, river, well and marine water. This chapter will consider current gold standard culture methods of analysis for E. coli and compare them with molecular DNA procedures. Established culture methods use β‐D-glucuronidase to identify E. coli and β‐D-galactosidase to detect coliforms. Emphasis will be placed on newer procedures that can be used onsite supported by laboratory procedures used for confirmation. Available rapid fluorimetric procedures which have been developed for use in the field, based on the assay of β‐D-glucuronidase, will be discussed. The rapid advances in procedures using a molecular approach will be considered and compared with the more established methods for determining E. coli in water. It is essential that all these methods should be quantitative in order to comply with legal norms, and in this regard, the potential involvement of biosensor technology will be of great value in successfully transferring laboratory procedures to the field

Wastewater monitoring for detection of public health markers during the COVID-19 pandemic: Near-source monitoring of schools in England over an academic year

BACKGROUND: Schools are high-risk settings for infectious disease transmission. Wastewater monitoring for infectious diseases has been used to identify and mitigate outbreaks in many near-source settings during the COVID-19 pandemic, including universities and hospitals but less is known about the technology when applied for school health protection. This study aimed to implement a wastewater surveillance system to detect SARS-CoV-2 and other public health markers from wastewater in schools in England. METHODS: A total of 855 wastewater samples were collected from 16 schools (10 primary, 5 secondary and 1 post-16 and further education) over 10 months of school term time. Wastewater was analysed for SARS-CoV-2 genomic copies of N1 and E genes by RT-qPCR. A subset of wastewater samples was sent for genomic sequencing, enabling determination of the presence of SARS-CoV-2 and emergence of variant(s) contributing to COVID-19 infections within schools. In total, >280 microbial pathogens and >1200 AMR genes were screened using RT-qPCR and metagenomics to consider the utility of these additional targets to further inform on health threats within the schools. RESULTS: We report on wastewater-based surveillance for COVID-19 within English primary, secondary and further education schools over a full academic year (October 2020 to July 2021). The highest positivity rate (80.4%) was observed in the week commencing 30th November 2020 during the emergence of the Alpha variant, indicating most schools contained people who were shedding the virus. There was high SARS-CoV-2 amplicon concentration (up to 9.2x106 GC/L) detected over the summer term (8th June - 6th July 2021) during Delta variant prevalence. The summer increase of SARS-CoV-2 in school wastewater was reflected in age-specific clinical COVID-19 cases. Alpha variant and Delta variant were identified in the wastewater by sequencing of samples collected from December to March and June to July, respectively. Lead/lag analysis between SARS-CoV-2 concentrations in school and WWTP data sets show a maximum correlation between the two-time series when school data are lagged by two weeks. Furthermore, wastewater sample enrichment coupled with metagenomic sequencing and rapid informatics enabled the detection of other clinically relevant viral and bacterial pathogens and AMR. CONCLUSIONS: Passive wastewater monitoring surveillance in schools can identify cases of COVID-19. Samples can be sequenced to monitor for emerging and current variants of concern at the resolution of school catchments. Wastewater based monitoring for SARS-CoV-2 is a useful tool for SARS-CoV-2 passive surveillance and could be applied for case identification and containment, and mitigation in schools and other congregate settings with high risks of transmission. Wastewater monitoring enables public health authorities to develop targeted prevention and education programmes for hygiene measures within undertested communities across a broad range of use cases

Environmental waters and E. coli as a marker, including pathogenic and resistant strains

Good public health depends on regular monitoring of water quality as faecal contamination is a serious problem due to the potential for contracting disease. Bacterial contamination in water is measured using indicator organisms, notably Escherichia coli and Enterococci which are used as primary indicators of contamination in fresh and marine water quality, respectively, rather than the total coliforms present. Although most E. coli and Enterococci strains cause only mild infections, their presence is indicative of the potential presence of other more pathogenic organisms which are a danger to human health. The acceptable levels of indicator organisms are defined in legislation and are set for drinking, river, well and marine water. This chapter will consider current gold standard culture methods of analysis for E. coli and compare them with molecular DNA procedures. Established culture methods use β‐D-glucuronidase to identify E. coli and β‐D-galactosidase to detect coliforms. Emphasis will be placed on newer procedures that can be used onsite supported by laboratory procedures used for confirmation. Available rapid fluorimetric procedures which have been developed for use in the field, based on the assay of β‐D-glucuronidase, will be discussed. The rapid advances in procedures using a molecular approach will be considered and compared with the more established methods for determining E. coli in water. It is essential that all these methods should be quantitative in order to comply with legal norms, and in this regard, the potential involvement of biosensor technology will be of great value in successfully transferring laboratory procedures to the field

Characterization of bacterial proteases with a panel of fluorescent peptide substrates

Bacteria produce a range of proteolytic enzymes. In an attempt to detect and identify bacteria on the basis of their protease activity, a panel of protease substrates was investigated. Peptides conjugated to the fluorophore 7-amino-4-methylcoumarin (AMC) are well-established substrates for measuring protease activity. Although peptide–AMC substrates are generally not specific for a single protease, a unique pattern can be achieved for both highly specific enzymes and those with a broader substrate range by comparing different peptide substrates. The panel of 7 peptide–AMC substrates chosen exhibited a unique pattern for nine microbial proteases. The selected peptides were used to determine protease activity in cultured strains of Pseudomonas aeruginosa and Staphylococcus aureus. A signal pattern obtained with peptides with arginine, lysine, and tyrosine in the P1 position characterized the bacterial protease activities in these samples. The kinetic parameters for the three best substrates for the P. aeruginosa sample were calculated. Further information about substrate specificity was gained by the selective use of protease inhibitors. The results presented show that peptide–AMC substrates provide a simple and sensitive tool to characterize protease activity in microbiological samples and that they have the potential to identify and distinguish different bacterial species