The efficacy of current treatment processes to remove, inactivate, or reduce environmental bloom-forming Escherichia coli.

Escherichia coli is excreted in high numbers from the intestinal tract of humans, other mammals, and birds. Traditionally, it had been thought that E. coli could grow only within human or animal hosts and would perish in the environment. Therefore, the presence of E. coli in water has become universally accepted as a key water quality indicator of fecal pollution. However, recent research challenges the assumption that the presence of E. coli in water is always an indicator of fecal contamination, with some types of E. coli having evolved to survive and grow in aquatic environments. These strains can form blooms in water storages, resulting in high E. coli counts even without fecal contamination. Although these bloom-forming strains lack virulence genes and pose little threat to public health, their presence in treated water triggers the same response as fecal-derived E. coli. Yet, little is known about the effectiveness of treatment processes in removing or inactivating them. This study evaluated the effectiveness of current treatment processes to remove bloom-forming strains, in comparison to fecal-derived strains, with conventional coagulation-flocculation-sedimentation and filtration investigated. Second, the effectiveness of current disinfection processes-chlorination, chloramination, and ultraviolet (UV) light to disinfect bloom-forming strains in comparison to fecal-derived strains-was assessed. These experiments showed that the responses of bloom isolates were not significantly different from those of fecal E. coli strains. Therefore, commonly used water treatment and disinfection processes are effective to remove bloom-forming E. coli strains from water.IMPORTANCEThe presence of Escherichia coli in water has long been used globally as a key indicator of fecal pollution and for quantifying water safety. Traditionally, it was believed that E. coli could only thrive within hosts and would perish outside, making its presence in water indicative of fecal contamination. However, recent research has unveiled strains of E. coli capable of surviving and proliferating in aquatic environments, forming blooms even in the absence of fecal contamination. While these bloom-forming strains lack the genes to be pathogenic, their detection in source or drinking water triggers the same response as fecal-derived E. coli. Yet, little is known about the efficacy of treatment processes in removing them. This study evaluated the effectiveness of conventional treatment and disinfection processes in removing bloom-forming strains compared to fecal-derived strains. Results indicate that these commonly used processes are equally effective against both types of E. coli, reassuring that bloom-forming E. coli strains can be eliminated from water.

Comparison of kits for SARS-CoV-2 extraction in liquid and passive samples.

Effective extraction and detection of viral nucleic acids from sewage are fundamental components of a successful SARS-CoV-2 sewage surveillance programme. As there is no standard method employed in sewage surveillance, understanding the performance of different extraction kits in the recovery of SARS-CoV-2 and the impact that PCR inhibitors have on quantification is essential to minimize data discrepancies caused by sample extraction. Three commercial nucleic acid extraction kits: the RNeasy PowerSoil Total RNA Kit (PS), the RNeasy PowerMicrobiome Kit (PMB), and the MagMAX™ Microbiome Ultra Nucleic Acid Isolation Kit (MM), with minor modifications, were evaluated. Their efficacy in recovering viral ribonucleic acid and removal of PCR inhibitors was assessed using two South Australian wastewater matrices-one from a major metropolitan site and one from a regional centre. Both had SARS-CoV-2 present due to active COVID-19 cases in these communities. Overall, the MM kit had a higher recovery of SARS-CoV-2 from the samples tested, followed by PMB and PS. The PMB kit performance was strongly influenced by the sample matrix when compared to the MM kit. It is recommended to assess the performance of extraction kits using different local wastewater matrices to ensure the accuracy and reliability of monitoring results to avoid false reporting.

The detection of Japanese encephalitis virus in municipal wastewater during an acute disease outbreak.

AIM: To demonstrate the capability of wastewater-based surveillance (WBS) as a tool for detecting potential cases of Japanese Encephalitis Virus (JEV) infection in the community. METHODS AND RESULTS: In this study, we explore the potential of WBS to detect cases of JEV infection by leveraging from an established SARS-CoV-2 wastewater surveillance program. We describe the use of two reverse transcriptase quantitative polymerase chain reaction (RTqPCR) assays targeting JEV to screen archived samples from two wastewater treatment plants (WWTPs). JEV was detected in wastewater samples collected during a timeframe coinciding with a cluster of acute human encephalitis cases, alongside concurrent evidence of JEV detection in mosquito surveillance and the sentinel chicken programs within South Australia's Riverland and Murraylands regions. CONCLUSIONS: Current surveillance measures for JEV encounter multiple constraints, which may miss the early stages of JEV circulation or fail to capture the full extent of transmission. The detection of JEV in wastewater during a disease outbreak highlights the potential WBS has as a complementary layer to existing monitoring efforts forming part of the One Health approach required for optimal disease response and control.

Comparison of in vitro growth characteristics of Cryptosporidium hominis (IdA15G1) and Cryptosporidium parvum (Iowa-IIaA17G2R1 and IIaA18G3R1).

Cryptosporidium is a major cause of diarrhoeal disease and mortality in young children in resource-poor countries, for which no vaccines or adequate therapeutic options are available. Infection in humans is primarily caused by two species: C. hominis and C. parvum. Despite C. hominis being the dominant species infecting humans in most countries, very little is known about its growth characteristics and life cycle in vitro, given that the majority of our knowledge of the in vitro development of Cryptosporidium has been based on C. parvum. In the present study, the growth and development of two C. parvum isolates (subtypes Iowa-IIaA17G2R1 and IIaA18G3R1) and one C. hominis isolate (subtype IdA15G1) in HCT-8 cells were examined and compared at 24 h and 48 h using morphological data acquired with scanning electron microscopy. Our data indicated no significant differences in the proportion of meronts or merozoites between species or subtypes at either time-point. Sexual development was observed at the 48-h time-point across both species through observations of both microgamonts and macrogamonts, with a higher frequency of macrogamont observations in C. hominis (IdA15G1) cultures at 48-h post-infection compared to both C. parvum subtypes. This corresponded to differences in the proportion of trophozoites observed at the same time point. No differences in proportion of microgamonts were observed between the three subtypes, which were rarely observed across all cultures. In summary, our data indicate that asexual development of C. hominis is similar to that of C. parvum, while sexual development is accelerated in C. hominis. This study provides new insights into differences in the in vitro growth characteristics of C. hominis when compared to C. parvum, which will facilitate our understanding of the sexual development of both species.

Sphingopyxis Species Isolated from Sand Filter Biofilm at an Australian Drinking Water Treatment Works.

Three strains isolated by geosmin enrichment from a sand filter in an Australian drinking water treatment works were genome sequenced to identify their taxonomic placement, and a bench-scale batch experiment confirmed their geosmin-degrading capability. Using the average nucleotide identity based on the MUMmer algorithm (ANIm), pairwise digital DNA-DNA hybridization (dDDH), and phylogenomic analyses, the strains were identified as Sphingopyxis species.

Proposed taxonomic revision of Giardia duodenalis.

Giardia duodenalis, Giardia enterica, Giardia intestinalis and Giardia lamblia are the synonyms for a species complex of 8-11 phylogenetically distinct species of Giardia infecting a broad range of animals including humans. Retrospective alignment of 8409 gene sequences from 3 loci confirmed host associations of Assemblages and sub-Assemblages within this species complex and molecular species delimitation testing confirmed that the Assemblages and sub-Assemblages AI and AII should be recognised as distinct species. It is recommended to synonymise the Assemblages with historic species descriptions based on host associations and consider descriptions for new species where no corresponding description exists. Synonyms, Giardia duodenalis, Giardia intestinalis and Giardia enterica, to be removed from synonymy: synonymise "Giardia duodenalis-Assemblage AI" syn. n. to Giardia duodenalis (Davaine, 1875), Kofoid and Christansen, 1915, synonymise "Giardia duodenalis-Assemblage AII" syn. n. to Giardia intestinalis (Lambl, 1859; Blanchard, 1885), Alexeieff, 1914 and synonymise "Giardia duodenalis-Assemblage B" syn. n. to Giardia enterica (Grassi, 1881), Kofoid, 1920. Host specific Assemblages synonymised: synonymise canid-associated "Giardia duodenalis-Assemblage C" syn. n. to Giardia canisHegner, 1922; synonymise artiodactyl-associated "Giardia duodenalis-Assemblage E" syn. n. to Giardia bovisFantham, 1921; synonymise feline-associated "Giardia duodenalis-Assemblage F" syn. n. to Giardia catiDeschiens, 1925; and synonymise rodent-associated "Giardia duodenalis-Assemblage G" syn. n. to Giardia simoniLavier, 1924. New description for parasite type infecting specific host: canid-associated "Giardia duodenalis-Assemblage D" named Giardia lupus, sp. n. (LSID: urn:lsid:zoobank.org:act:1651A8CB-CBA8-40D9-AB59-D4AB11AC18A3). New proposed names and descriptions for consideration for parasite types infecting specific hosts: cervid-associated "Giardia duodenalis-sub-Assemblage AIII" for consideration "cervus" and Pinnipedia-associated "Giardia duodenalis-Assemblage H" for consideration "pinnipedis".

Stormwater monitoring using on-line UV-Vis spectroscopy.

Stormwater runoff contains a myriad of pollutants, including faecal microbes, and can pose a threat to urban water supplies, impacting both economic development and public health. Therefore, it is a necessity to implement a real-time hazard detection system that can collect a substantial amount of data, assisting water authorities to develop preventive strategies to ensure the control of hazards entering drinking water sources. An on-line UV-Vis spectrophotometer was applied in the field to collect real-time continuous data for various water quality parameters (nitrate, DOC, turbidity and total suspended solids) during three storm events in Mannum, Adelaide, Australia. This study demonstrated that the trends for on-line and comparative laboratory-analysed samples were complimentary through the events. Nitrate and DOC showed a negative correlation with water level, while turbidity and total suspended solids indicated a positive correlation with water level during the high rainfall intensity. The correlations among nitrate, DOC, turbidity, total suspended solids and water level are the opposite during low rainfall intensity. Nitrate, one of the main pollutants in stormwater, was investigated and used as a surrogate parameter for microbial detection. However, the microbiological data (Escherichia coli) from captured storm events showed poor correlations to nitrate and other typical on-line parameters in this study. This is possibly explained by the nature of the stormwater catchment outside of rain events, where the sources of bacteria and nutrients may be physically separated until mixed during surface runoff as a result of rainfall. In addition, the poor correlations among the microbiological data and on-line parameters could be due to the different sources of bacteria and nutrients that were transported to the stormwater drain where sampling and measurement were conducted.

Inactivation, removal, and regrowth potential of opportunistic pathogens and antimicrobial resistance genes in recycled water systems.

With two thirds of the global population living in areas affected by water scarcity, wastewater reuse is actively being implemented or explored by many nations. There is a need to better understand the efficacy of recycled water treatment plants (RWTPs) for removal of human opportunistic pathogens and antimicrobial resistant microorganisms. Here, we used a suite of probe-based multiplex and SYBR green real-time PCR assays to monitor enteric opportunistic pathogens (EOPs; Acinetobacter baumannii, Arcobacter butzlieri, Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, Legionella spp., Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella Enteritidis, Streptococcus spp.) and antimicrobial resistance genes (ARGs; qnrS, blaSHV, blaTEM, blaGES, blaKPC, blaIMI, blaSME, blaNDM, blaVIM, blaIMP, blaOXA-48-like, mcr-1 and mcr-3) of key concern from an antimicrobial resistance (AMR), waterborne and foodborne disease perspective. The class 1 integron-integrase gene (intl1) was quantified as a proxy for multi-drug resistance. EOPs, intl1 and ARGs absolute abundance (DNA and RNA) and metabolic activity (RNA) was assessed through three RWTPs with differing treatment trains. Our results indicate that RWTPs produced high quality recycled water for non-potable reuse by removing >95% of EOPs and ARGs, however, subpopulations of EOPs and ARGs survived disinfection and demonstrated potential to become actively growing members of the recycled water and distribution system microbiomes. The persistence of functional intl1 suggests that significant genetic recombination capacity remains in the recycled water, along with the likely presence of multi-drug resistant bacteria. Results provide new insights into the persistence and growth of EOPs, and prevalence and removal of ARGs in recycled water systems. These data will contribute towards the emerging evidence base of AMR risks in recycled water to inform quantitative risk-based policy development regarding water recycling schemes.

Wastewater-based epidemiology-surveillance and early detection of waterborne pathogens with a focus on SARS-CoV-2, Cryptosporidium and Giardia.

Waterborne diseases are a major global problem, resulting in high morbidity and mortality, and massive economic costs. The ability to rapidly and reliably detect and monitor the spread of waterborne diseases is vital for early intervention and preventing more widespread disease outbreaks. Pathogens are, however, difficult to detect in water and are not practicably detectable at acceptable concentrations that need to be achieved in treated drinking water (which are of the order one per million litre). Furthermore, current clinical-based surveillance methods have many limitations such as the invasive nature of the testing and the challenges in testing large numbers of people. Wastewater-based epidemiology (WBE), which is based on the analysis of wastewater to monitor the emergence and spread of infectious disease at a population level, has received renewed attention in light of the current coronavirus disease 2019 (COVID-19) pandemic. The present review will focus on the application of WBE for the detection and surveillance of pathogens with a focus on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the waterborne protozoan parasites Cryptosporidium and Giardia. The review highlights the benefits and challenges of WBE and the future of this tool for community-wide infectious disease surveillance.

Organoids and Bioengineered Intestinal Models: Potential Solutions to the Cryptosporidium Culturing Dilemma.

Cryptosporidium is a major cause of severe diarrhea-related disease in children in developing countries, but currently no vaccine or effective treatment exists for those who are most at risk of serious illness. This is partly due to the lack of in vitro culturing methods that are able to support the entire Cryptosporidium life cycle, which has led to research in Cryptosporidium biology lagging behind other protozoan parasites. In vivo models such as gnotobiotic piglets are complex, and standard in vitro culturing methods in transformed cell lines, such as HCT-8 cells, have not been able to fully support fertilization occurring in vitro. Additionally, the Cryptosporidium life cycle has also been reported to occur in the absence of host cells. Recently developed bioengineered intestinal models, however, have shown more promising results and are able to reproduce a whole cycle of infectivity in one model system. This review evaluates the recent advances in Cryptosporidium culturing techniques and proposes future directions for research that may build upon these successes.

Toolbox for the sampling and monitoring of benthic cyanobacteria.

Benthic cyanobacteria are a nuisance because they produce highly potent toxins and taste and odour compounds. Despite this, benthic cyanobacteria remain far less studied than their planktonic counterparts. For example, little is known about their growth or the seasonality of their secondary metabolite production. Moreover, sampling and monitoring techniques commonly used for the survey of planktonic species are not necessarily applicable to benthic forms. This study aimed to develop and validate a new sampling device for the routine monitoring of benthic mats. Molecular monitoring techniques were established and validated on environmental samples collected in a South Australian reservoir (SA-L2). A total of eight qPCR assays were applied to samples in order to track seasonal variations in cyanobacteria concentrations and associated secondary metabolite production. Next Generation Sequencing was utilised to conduct a microbial community composition analysis and to select the most appropriate substrate material for the sampling of benthic cyanobacteria. The concentration of the secondary metabolites geosmin and 2-methyl-isoborneol were quantified using High-Performance Liquid Chromatography, and concentrations of key nutrients (N, P) were quantified in water samples. The sampling device designed proved efficient and easy to use in the field. The qPCR assay designed for the amplification of the cyanobacterial MIB synthase had a high efficiency with a minimum limit of quantification of 4 cell-equivalents per reaction and identified a potential source of MIB in SA-L2 Reservoir. The peak season for benthic growth and secondary metabolite production was observed in spring. Proportionally, 35% of the variability in water geosmin concentrations can be explained by benthic actinobacterial and cyanobacterial activity, showing that freshwater benthic mats represent a significant source of taste and odour compounds.

Field based pilot-scale drinking water distribution system: Simulation of long hydraulic retention times and microbiological mediated monochloramine decay.

Drinking water distribution systems with long hydraulic retention times (HRTs) commonly encounter rapid microbiological-mediated monochloramine decay that results in microbial regrowth and nitrification with reduction in alkalinity. In this paper, we report the design and operation of a field-based pilot-scale distribution system (PDS) operated at flows that simulate long HRTs (∼10 days) to promote rapid microbiological monochloramine decay over long periods. The PDS is designed to enable the testing of chemical treatment for the control of nitrification and monochloramine decay. The PDS has two identical cylindrical polyethylene tanks (DS1 & DS2), each of 1 m diameter and 1.8 m height (∼1 kL) holding 900 m of polyethylene (PE) tubing with sampling points every 300 m intervals. Microbial mediated decay (determined by the Fm test) of monochloramine occurred as treated (alum coagulated and flocculated) and chloraminated water passed through the DSs. In this manuscript we report: •An inexpensive, flexible and compact system that can be readily set-up at a full-scale water treatment plant, requiring minimal supervision for operation.•A 'draw & fill' system for achieving control on HRT's through the pipes.

Understanding human infectious Cryptosporidium risk in drinking water supply catchments.

Treating drinking water appropriately depends, in part, on the robustness of source water quality risk assessments, however quantifying the proportion of infectious, human pathogenic Cryptosporidium oocysts remains a significant challenge. We analysed 962 source water samples across nine locations to profile the occurrence, rate and timing of infectious, human pathogenic Cryptosporidium in surface waters entering drinking water reservoirs during rainfall-runoff conditions. At the catchment level, average infectivity over the four-year study period reached 18%; however, most locations averaged <5%. The maximum recorded infectivity fraction within a single rainfall runoff event was 65.4%, and was dominated by C. parvum. Twenty-two Cryptosporidium species and genotypes were identified using PCR-based molecular techniques; the most common being C. parvum, detected in 23% of water samples. Associations between landuse and livestock stocking characteristics with Cryptosporidium were determined using a linear mixed-effects model. The concentration of pathogens in water were significantly influenced by flow and dominance of land-use by commercial grazing properties (as opposed to lifestyle properties) in the catchment (p < 0.01). Inclusion of measured infectivity and human pathogenicity data into a quantitative microbial risk assessment (QMRA) could reduce the source water treatment requirements by up to 2.67 log removal values, depending on the catchment, and demonstrated the potential benefit of collating such data for QMRAs.

Cryptosporidium species and subtypes in animals inhabiting drinking water catchments in three states across Australia.

As part of long-term monitoring of Cryptosporidium in water catchments serving Western Australia, New South Wales (Sydney) and Queensland, Australia, we characterised Cryptosporidium in a total of 5774 faecal samples from 17 known host species and 7 unknown bird samples, in 11 water catchment areas over a period of 30 months (July 2013 to December 2015). All samples were initially screened for Cryptosporidium spp. at the 18S rRNA locus using a quantitative PCR (qPCR). Positives samples were then typed by sequence analysis of an 825 bp fragment of the 18S gene and subtyped at the glycoprotein 60 (gp60) locus (832 bp). The overall prevalence of Cryptosporidium across the various hosts sampled was 18.3% (1054/5774; 95% CI, 17.3-19.3). Of these, 873 samples produced clean Sanger sequencing chromatograms, and the remaining 181 samples, which initially produced chromatograms suggesting the presence of multiple different sequences, were re-analysed by Next- Generation Sequencing (NGS) to resolve the presence of Cryptosporidium and the species composition of potential mixed infections. The overall prevalence of confirmed mixed infection was 1.7% (98/5774), and in the remaining 83 samples, NGS only detected one species of Cryptosporidium. Of the 17 Cryptosporidium species and four genotypes detected (Sanger sequencing combined with NGS), 13 are capable of infecting humans; C. parvum, C. hominis, C. ubiquitum, C. cuniculus, C. meleagridis, C. canis, C. felis, C. muris, C. suis, C. scrofarum, C. bovis, C. erinacei and C. fayeri. Oocyst numbers per gram of faeces (g-1) were also determined using qPCR, with medians varying from 6021-61,064 across the three states. The significant findings were the detection of C. hominis in cattle and kangaroo faeces and the high prevalence of C. parvum in cattle. In addition, two novel C. fayeri subtypes (IVaA11G3T1 and IVgA10G1T1R1) and one novel C. meleagridis subtype (IIIeA18G2R1) were identified. This is also the first report of C. erinacei in Australia. Future work to monitor the prevalence of Cryptosporidium species and subtypes in animals in these catchments is warranted.

Profiling the diversity of Cryptosporidium species and genotypes in wastewater treatment plants in Australia using next generation sequencing.

Wastewater recycling is an increasingly popular option in worldwide to reduce pressure on water supplies due to population growth and climate change. Cryptosporidium spp. are among the most common parasites found in wastewater and understanding the prevalence of human-infectious species is essential for accurate quantitative microbial risk assessment (QMRA) and cost-effective management of wastewater. The present study conducted next generation sequencing (NGS) to determine the prevalence and diversity of Cryptosporidium species in 730 raw influent samples from 25 Australian wastewater treatment plants (WWTPs) across three states: New South Wales (NSW), Queensland (QLD) and Western Australia (WA), between 2014 and 2015. All samples were screened for the presence of Cryptosporidium at the 18S rRNA (18S) locus using quantitative PCR (qPCR), oocyst numbers were determined directly from the qPCR data using DNA standards calibrated by droplet digital PCR, and positives were characterized using NGS of 18S amplicons. Positives were also screened using C. parvum and C. hominis specific qPCRs. The overall Cryptosporidium prevalence was 11.4% (83/730): 14.3% (3/21) in NSW; 10.8% (51/470) in QLD; and 12.1% (29/239) in WA. A total of 17 Cryptosporidium species and six genotypes were detected by NGS. In NSW, C. hominis and Cryptosporidium rat genotype III were the most prevalent species (9.5% each). In QLD, C. galli, C. muris and C. parvum were the three most prevalent species (7.7%, 5.7%, and 4.5%, respectively), while in WA, C. meleagridis was the most prevalent species (6.3%). The oocyst load/Litre ranged from 70 to 18,055 oocysts/L (overall mean of 3426 oocysts/L: 4746 oocysts/L in NSW; 3578 oocysts/L in QLD; and 3292 oocysts/L in WA). NGS-based profiling demonstrated that Cryptosporidium is prevalent in the raw influent across Australia and revealed a large diversity of Cryptosporidium species and genotypes, which indicates the potential contribution of livestock, wildlife and birds to wastewater contamination.

Evaluating membrane performance in recycled water treatment plants for assets replacement strategy.

Membranes are an important barrier used in recycled water treatment plants for pathogen removal. Understanding performance over operational life is important to inform membrane replacement. In this study, full scale virus challenge testing was conducted on newly commissioned membranes to validate virus log removal values for accreditation. After six years of operation, the membrane integrity was repeated to ensure compliance with the state regulatory health authority and gain an understanding of the asset's condition. Membrane performance was assessed using a combination of complementary tests including membrane autopsy and chemical tolerance testing to assess individual modules and selected membrane fibres, followed by a full scale virus challenge for whole of unit assessment. The results demonstrated that the aged membrane fibres were intact and had not been affected by long-term exposure to chlorine, which provides valuable information for membrane asset replacement strategies.

Benthic cyanobacteria: A source of cylindrospermopsin and microcystin in Australian drinking water reservoirs.

Cyanobacteria represent a health hazard worldwide due to their production of a range of highly potent toxins in diverse aquatic environments. While planktonic species have been the subject of many investigations in terms of risk assessment, little is known about benthic forms and their impact on water quality or human and animal health. This study aimed to purify isolates from environmental benthic biofilms sampled from three different drinking water reservoirs and to assess their toxin production by using the following methods: Enzyme-Linked Immunosorbent Assay (ELISA), High-Performance Liquid Chromatography (HPLC), Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) and quantitative PCR (qPCR). Microscopic observation of the isolates allowed the identification of various filamentous cyanobacterial genera: Anabaena (benthic form), Calothrix and Nostoc from the Nostocales and Geitlerinema, Leptolyngbya, Limnothrix, Lyngbya, Oxynema, Phormidium and Pseudanabaena representing non-heterocystous filamentous cyanobacteria. The Phormidium ambiguum strain AWQC-PHO021 was found to produce 739 ng/mg of dry weight (d/w) of cylindrospermopsin and 107 ng/mg (d/w) of deoxy-cylindrospermopsin. The Nostoc linckia strain AWQC-NOS001 produced 400 ng/mg (d/w) of a microcystin analogue. This is the first report of hepatotoxin production by benthic cyanobacteria in temperate Australian drinking water reservoirs. These findings indicate that water quality monitoring programs need to consider benthic cyanobacteria as a potential source of toxins.

Virus removal by ultrafiltration: Understanding long-term performance change by application of Bayesian analysis.

Ultrafiltration is an effective barrier to waterborne pathogens including viruses. Challenge testing is commonly used to test the inherent reliability of such systems. Performance validation seeks to demonstrate the adequate reliability of the treatment system. Appropriate and rigorous data analysis is an essential aspect of validation testing. In this study we used Bayesian analysis to assess the performance of a full-scale ultrafiltration system which was validated and revalidated after five years of operation. A hierarchical Bayesian model was used to analyse a number of similar ultrafiltration membrane skids working in parallel during the two validation periods. This approach enhanced our ability to obtain accurate estimations of performance variability, especially when the sample size of some system skids was limited. This methodology enabled the quantitative estimation of uncertainty in the performance parameters and generation of predictive distributions incorporating those uncertainties. The results indicated that there was a decrease in the mean skid performance after five years of operation of approximately 1 log reduction value (LRV). Interestingly, variability in the LRV also reduced, with standard deviations from the revalidation data being decreased by a mean 0.37 LRV compared with the original validation data. The model was also useful in comparing the operating performance of the various parallel skids within the same year. Evidence of differences was obtained in 2015 for one of the membrane skids. A hierarchical Bayesian analysis of validation data provides robust estimations of performance and the incorporation of probabilistic analysis which is increasingly important for comprehensive quantitative risk assessment purposes.

Novel Primer Sets for Next Generation Sequencing-Based Analyses of Water Quality.

Next generation sequencing (NGS) has rapidly become an invaluable tool for the detection, identification and relative quantification of environmental microorganisms. Here, we demonstrate two new 16S rDNA primer sets, which are compatible with NGS approaches and are primarily for use in water quality studies. Compared to 16S rRNA gene based universal primers, in silico and experimental analyses demonstrated that the new primers showed increased specificity for the Cyanobacteria and Proteobacteria phyla, allowing increased sensitivity for the detection, identification and relative quantification of toxic bloom-forming microalgae, microbial water quality bioindicators and common pathogens. Significantly, Cyanobacterial and Proteobacterial sequences accounted for ca. 95% of all sequences obtained within NGS runs (when compared to ca. 50% with standard universal NGS primers), providing higher sensitivity and greater phylogenetic resolution of key water quality microbial groups. The increased selectivity of the new primers allow the parallel sequencing of more samples through reduced sequence retrieval levels required to detect target groups, potentially reducing NGS costs by 50% but still guaranteeing optimal coverage and species discrimination.