F-RNA Phages Research Articles

The toxicity of cationic surfactant HDTMA-Br, desorbed from surfactant modified zeolite, towards faecal indicator and environmental microorganisms

Surfactant Modified Zeolite (SMZ) represents a versatile, cost-effective permeable reactive material, capable of treating multiple classes of contaminants. The potential for HDTMA-Br, a cationic surfactant commonly used to modify zeolite, to desorb from the zeolite surface has been identified as a potential issue for the ongoing use of SMZ in water remediation contexts. This paper investigates the toxicity of HDTMA-Br towards enteric virus surrogates, F-RNA bacteriophage MS2 and E. coli, Bacillus subtilis, and soil microflora. The concentration of surfactant desorbing from SMZ was quantified through a bioassay using E. coli.Results showed HDTMA-Br concentrations of ≥10−5M were toxic to MS2, ≥10−4M were toxic to E. coli and ≥10−6M were toxic to B. subtilis. No toxic relationship was established between HDTMA-Br and soil microflora. Desorption of ≥10−4M of HDTMA-Br was shown for the two SMZ samples under the mixing conditions used. Effects of this surfactant on total soil microflora were ambiguous since no toxic relationship could be established, however, HDTMA-Br, at concentrations desorbing from SMZ, were shown to impact the soil bacterium B. subtilis. Further research is required to determine the effect of this surfactant on microbial populations and species diversity in soils.

FRNA Bacteriophages as Viral Indicators of Faecal Contamination in Mexican Tropical Aquatic Systems

A particular challenge to water safety in populous intertropical regions is the lack of reliable faecal indicators to detect microbiological contamination of water, while the numerical relationships of specific viral indicators remain largely unexplored. The aim of this study was to investigate the numerical relationships of FRNA-bacteriophage genotypes, adenovirus 41, and human adenoviruses (HADV) in Mexican surface water systems to assess sewage contamination. We studied the presence of HADV, HADV41 and FRNA bacteriophage genotypes in water samples and quantified by qPCR and RT-qPCR. Virus and water quality indicator variances, as analyzed by principal component analysis and partial least squared regression, followed along the major percentiles of water faecal enterococci. FRNA bacteriophages adequately deciphered viral and point source water contamination. The strongest correlation for HADV was with FRNA bacteriophage type II, in water samples higher than the 50th percentiles of faecal enterococci, thus indicating urban pollution. FRNA bacteriophage genotypes I and III virus indicator performances were assisted by their associations with electrical conductivity and faecal enterococci. In combination, our methods are useful for inferring water quality degradation caused by sewage contamination. The methods used have potential for determining source contamination in water and, specifically, the presence of enteric viruses where clean and contaminated water have mixed.

Bioaccumulation and persistence of faecal bacterial and viral indicators in Mytilus edulis and Crassostrea gigas.

This study investigated the response of two shellfish species - mussels (Mytilus edulis) and oysters (Crassostrea gigas) to microbial contamination in order to gain a better understanding of the bioaccumulation and persistence of microorganisms under controlled conditions. M. edulis and C. gigas were placed in sterile laboratory-prepared artificial seawater and initial tests were carried out to ensure both faecal indicator bacteria (FIB) and bacteriophages were below detection limits. FIB and phages were isolated, purified and dosed into experimental tanks containing the shellfish species. The GB124 phages were bioaccumulated to the highest concentration in M. edulis (1.88Log10) and C. gigas (1.27Log10) after 24h. Somatic coliphages were bioaccumulated to the highest concentration in both M. edulis (4.84log10) and C. gigas (1.73log10) after 48h. F-RNA phages were bioaccumulated to the highest concentration in M. edulis (3.51log10) after 6h but were below detection limit in C. gigas throughout the exposure period. Escherichia coli, faecal coliforms and intestinal enterococci were bioaccumulated to the highest concentrations in M. edulis (5.05log10, 5.06log10 and 3.98log10, respectively) after 48h. In C. gigas, E. coli reached a maximum concentration (5.47log10) after 6h, faecal coliforms (5.19log10) after 12h and intestinal enterococci (3.23log10) after 24h.M. edulis bioaccumulated phages to a greater extent than the faecal bacteria, and in both shellfish species, faecal bacteria persisted for longer periods over 48h than the phages. This study highlights significant variation in the levels and rate of accumulation and persistence with respect to both shellfish species and the indicators used to assess risk. The results suggest that phage-based indicators could help elucidate risks to human health associated with pathogenic organisms.

Fecal Bacteria, Bacteriophage, and Nutrient Reductions in a Full-Scale Denitrifying Woodchip Bioreactor.

Denitrifying bioreactors using woodchips or other slow-release carbon sources can be an effective method for removing nitrate (NO) from wastewater and tile drainage. However, the ability of these systems to remove fecal microbes from wastewater has been largely uninvestigated. In this study, reductions in fecal indicator bacteria () and viruses (F-specific RNA bacteriophage [FRNA phage]) were analyzed by monthly sampling along a longitudinal transect within a full-scale denitrifying woodchip bioreactor receiving secondary-treated septic tank effluent. Nitrogen, phosphorus, 5-d carbonaceous biochemical oxygen demand (CBOD), and total suspended solids (TSS) reduction were also assessed. The bioreactor demonstrated consistent and substantial reduction of (2.9 log reduction) and FRNA phage (3.9 log reduction) despite receiving highly fluctuating inflow concentrations [up to 3.5 × 10 MPN (100 mL) and 1.1 × 10 plaque-forming units (100 mL) , respectively]. Most of the removal of fecal microbial contaminants occurred within the first meter of the system (1.4 log reduction for ; 1.8 log reduction for FRNA phage). The system was also efficient at removing NO (>99.9% reduction) and TSS (89% reduction). There was no evidence of consistent removal of ammonium, organic nitrogen, or phosphorus. Leaching of CBOD occurred during initial operation but decreased and stabilized at lower values (14 g O m) after 9 mo. We present strong evidence for reliable microbial contaminant removal in denitrifying bioreactors, demonstrating their broader versatility for wastewater treatment. Research on the removal mechanisms of microbial contaminants in these systems, together with the assessment of longevity of removal, is warranted.

Hazardous events in membrane bioreactors – Part 3: Impacts on microorganism log removal efficiencies

Under normal operation, membrane bioreactors (MBRs) produce high quality effluent leading to common application in water recycling schemes. When hazardous events, outside of normal operation, occur strategies must be in place to ensure that microbially safe water is consistently produced. Limited literature is available to quantify the consequence of hazardous events on microorganism log removal value (LRV) by MBRs. As a result, current MBR risk management practices cannot be based on sufficient quantitative evidence. In this study, MBRs were subjected to hazardous events designed to reflect a range of potential severe feed water variations and process failures. During these challenge events, removal of four microbial indicator organisms representing viruses, bacteria and protozoa were quantified. Hazardous event impacts were benchmarked against the 5th percentile of normal process LRV, determined via probabilistic techniques. Severe feed water variation decreased the efficacy of bio-predation due to adversely affected activated sludge performance. However, overall LRV was preserved due to the onset of fouling, aiding rejection by the membrane. Overall FRNA bacteriophage LRV significantly decreased to 0.3log units below the 5th percentile (LRV=4.1) after the NaCl shock, most likely due to inhibition of adsorption mechanisms. Fibre breakage resulted in a significant reduction in LRV, but was mitigated by suspended solids plugging the membrane lumen within 15min. The findings presented here can inform risk management strategies for MBRs by permitting control strategy prioritisation according to quantified event consequences.

Modelling pathogen log10 reduction values achieved by activated sludge treatment using naïve and semi naïve Bayes network models

Risk management for wastewater treatment and reuse have led to growing interest in understanding and optimising pathogen reduction during biological treatment processes. However, modelling pathogen reduction is often limited by poor characterization of the relationships between variables and incomplete knowledge of removal mechanisms. The aim of this paper was to assess the applicability of Bayesian belief network models to represent associations between pathogen reduction, and operating conditions and monitoring parameters and predict AS performance. Naïve Bayes and semi-naïve Bayes networks were constructed from an activated sludge dataset including operating and monitoring parameters, and removal efficiencies for two pathogens (native Giardia lamblia and seeded Cryptosporidium parvum) and five native microbial indicators (F-RNA bacteriophage, Clostridium perfringens, Escherichia coli, coliforms and enterococci). First we defined the Bayesian network structures for the two pathogen log10 reduction values (LRVs) class nodes discretized into two states (< and ≥1 LRV) using two different learning algorithms. Eight metrics, such as Prediction Accuracy (PA) and Area Under the receiver operating Curve (AUC), provided a comparison of model prediction performance, certainty and goodness of fit. This comparison was used to select the optimum models. The optimum Tree Augmented naïve models predicted removal efficiency with high AUC when all system parameters were used simultaneously (AUCs for C. parvum and G. lamblia LRVs of 0.95 and 0.87 respectively). However, metrics for individual system parameters showed only the C. parvum model was reliable. By contrast individual parameters for G. lamblia LRV prediction typically obtained low AUC scores (AUC < 0.81). Useful predictors for C. parvum LRV included solids retention time, turbidity and total coliform LRV. The methodology developed appears applicable for predicting pathogen removal efficiency in water treatment systems generally.

Enumeration of Somatic and F-RNA Phages as an Indicator of Fecal Contamination in Potable Water from Rural Areas of the North West Province.

Bacteriophages are regarded as enteric viral indicators in faecally contaminated water systems and may indicate the presence of human viral pollution. They are relatively resistant to inactivation by natural and treatment processes. In this study, the presence of somatic coliphages and F-RNA coliphages was investigated in potable water from rural areas in the North West province. Water samples were aseptically collected from boreholes and tap water from some rural communities in the North West Province. Physical parameters of the water, such as the temperature, pH and turbidity, were measured before sample collection. Double-agar layer assay was performed using ISO, (1995, 2000) standard methods. Bottled water was used as a negative control and the strains фX174 and MS2 as positive controls. Of the 16 water samples collected, 15 were positive for somatic bacteriophages while F-RNA coliphages were detected in only two samples. Amongst the positive samples 189 and three plaque forming units were obtained for both somatic and F-RNA coliphages, respectively. No coliphage was detected in water from Masamane tap 1. The rest of the samples obtained from various rural areas were positive and did not comply with national and international standards for potable water. This was a cause for concern and should be further investigated.

Detection and removal of pathogenic norovirus employing tertiary treatment in wastewater and water treatment facilities

Human pathogenic viruses (HPVs) with particular reference to norovirus (NoV) commonly known as ‘the winter vomiting bug’ is the leading causes of viral gastroenteritis worldwide. NoV is present in wastewater in high numbers and coupled with a low infectious dose can endanger public health. Secondary wastewater treatment processes are not designed to eradicate HPVs thus a tertiary disinfection step is required. Typical pathogen removal processes include slow sand filtration and membrane barrier methods. NoV cannot be cultured in a laboratory and so its sole detection is limited to expensive and time-consuming molecular methods. The use of an alternative virus, the FRNA bacteriophages has been suggested as a surrogate for NoV due to its physicochemical similarities and cultivability. This research focuses on the investigation of a barrier method of tertiary disinfection using membrane filters and tangential flow filtration. In addition to this, the suitability of FRNA bacteriophages as a cheaper alternative indicator of NoV was investigated.

The impact of major earthquakes and subsequent sewage discharges on the microbial quality of water and sediments in an urban river

A series of large earthquakes struck the city of Christchurch, New Zealand in 2010–2011. Major damage sustained by the sewerage infrastructure required direct discharge of up to 38,000m3/day of raw sewage into the Avon River of Christchurch for approximately six months. This allowed evaluation of the relationship between concentrations of indicator microorganisms (Escherichia coli, Clostridium perfringens and F-RNA phage) and pathogens (Campylobacter, Giardia and Cryptosporidium) in recreational water and sediment both during and post-cessation of sewage discharges.Giardia was the pathogen found most frequently in river water and sediment, although Campylobacter was found at higher levels in water samples. E. coli levels in water above 550CFU/100mL were associated with increased likelihood of detection of Campylobacter, Giardia and Cryptosporidium, supporting the use of E. coli as a reliable indicator for public health risk. The strength of the correlation of microbial indicators with pathogen detection in water decreased in the following order: E. coli>F-RNA phage>C. perfringens.All the microorganisms assayed in this study could be recovered from sediments. C. perfringens was observed to accumulate in sediments, which may have confounded its usefulness as an indicator of fresh sewage discharge. F-RNA phage, however, did not appear to accumulate in sediment and in conjunction with E. coli, may have potential as an indicator of recent human sewage discharge in freshwater. There is evidence to support the low-level persistence of Cryptosporidium and Giardia, but not Campylobacter, in river sediments after cessation of sewage discharges. In the event of disturbances of the sediment, it is highly probable that there could be re-mobilisation of microorganisms beyond the sediment–water exchange processes occurring under base flow conditions. Re-suspension events do, therefore, increase the potential risk to human health for those who participate in recreational and work-related activities in the river environment.

Numbers of coliforms, Escherichia coli, F-RNA phage, rotavirus, bovine enteric calicivirus and presence of non-O157 STEC on commercial vacuum packaged beef

The numbers of coliforms, Escherichia coli, F-RNA coliphages, bovine enteric calicivirus (BEC) and rotavirus (RV) and presence of non-O157 shiga toxigenic E. coli (STEC) were determined on commercial vacuum packaged beef subprimals at the retail level from swabs obtained from the entire surfaces of 150 cuts that originated from federally and provincially registered plants. The prevalence and log mean numbers of E. coli were higher in provincially registered plants than in federally registered plants; 64% vs 20%, respectively, and −0.3 vs −1.22 log cfu/100 cm2, respectively. In contrast, the prevalence and mean log numbers of F-RNA coliphages were lower for the provincially registered plants than for the federally registered plants; 31% vs 68% and −0.86 vs −0.13 log cfu/100 cm2, respectively. One E. coli sample tested positive for stx2 and eae. F-RNA coliphages associated with human origin (GII/GIII) were detected in 12% and 30% of samples that originated from provincially and federally registered plants, respectively. RV RNA was detected in 4% of samples while BEC RNA was not detected. Although the infectivity of RV is unknown, the presence of viable F-RNA coliphages suggests that consumers could potentially be at risk when consuming undercooked meat that is contaminated with RV.

Fluctuation of densities of bacteriophages and Escherichia coli present in natural biofilms and water of a main channel and a small tributary

Fluctuation of bacteriophage and Escherichia coli densities in naturally developed riverbed biofilms were investigated for a 1-year period. E. coli ranged from 1,500 to 15,500 most probable number (MPN)/100 mL and from 580 to 18,500 MPN/cm(2) in the main channel in the river water and biofilms, respectively. However, the fluctuations were much greater in the tributary, ranging from 0.8 to 100 MPN/100 mL and from 0.3 to 185 MPN/cm(2) in water and biofilms, respectively. The fluctuations of coliphages were also greater in the tributary than in the main channel. FRNA phage serotyping results indicated no significant differences in the source type of the fecal contamination in the main channel and tributary sampling stations. Significant correlations between phage groups in biofilms and water were found at both main channel and tributary. It was assumed that natural biofilms developed in the streambed captured and retained somatic phages in the biofilms for a certain period of time in the main channel site. At the location receiving constant and heavy contamination, the usage of phage indicators may provide additional information on the presence of viruses. In the small tributary it may be possible to estimate the virus concentration by monitoring the E. coli indicator.

Norovirus and FRNA bacteriophage determined by RT-qPCR and infectious FRNA bacteriophage in wastewater and oysters

Norovirus (NoV), the leading cause of adult non-bacterial gastroenteritis can be commonly detected in wastewater but the extent of NoV removal provided by wastewater treatment plants (WWTPs) is unclear. We monitored a newly commissioned WWTP with UV disinfection on a weekly basis over a six month period for NoV using RT-qPCR and for FRNA bacteriophage GA using both RT-qPCR (total concentration) and a plaque assay (infectious concentration). Mean concentrations of NoV GI and GII in influent wastewater were reduced by 0.25 and 0.41 log10 genome copies 100 ml−1, respectively by the WWTP. The mean concentration of total FRNA bacteriophage GA was reduced by 0.35 log genome copies 100 ml−1 compared to a reduction of infectious FRNA bacteriophage GA of 2.13 log PFU 100 ml−1. A significant difference between concentrations of infectious and total FRNA bacteriophage GA was observed in treated, but not in untreated wastewaters. We conclude that RT-qPCR in isolation underestimates the reduction of infectious virus during wastewater treatment. We further compared the concentrations of infectious virus in combined sewer overflow (CSO) and UV treated effluents using FRNA bacteriophage GA. A greater percentage (98%) of infectious virus is released in CSO discharges than UV treated effluent (44%). Following a CSO discharge, concentrations of NoV GII and infectious FRNA bacteriophage GA in oysters from less than the limit of detection to 3150 genome copies 100 g−1 and 1050 PFU 100 g−1 respectively.

Potential of Pulsed Light to Inactivate Bacteriophage MS2 in Simple Liquid Medium and on Complex Foodstuffs.

The virucidal efficacy of a pulsed light treatment applied to viral suspensions, glass beads and herb powders was studied for the F-RNA bacteriophage MS2. The experimental results obtained demonstrated the high potential of this technology to efficiently decontaminate simple matrices but underlined the complexity of application to complex food matrices.

Validation of activated sludge plant performance for virus and protozoan reduction

Validation studies were undertaken at Adelaide metropolitan wastewater treatment plants to establish the actual log10 reduction values (LRVs) of pathogens (viruses and Cryptosporidium) across activated sludge plants (ASPs) as an alternative to accepting the default values attributed by the Department of Health and Ageing (DHA). Grab samples were collected across a 6-week period and assessed for pathogens (adenovirus and Cryptosporidium) and indicator microorganisms (sulphite-reducing clostridia and F-RNA bacteriophage). Through applying the validation process, the DHA has revised the default value for reduction of viruses with an increase from 0.5 log10 to 1 log10 while the value for protozoa remains at 0.5 log10 based on the combined data for a well-operated and maintained ASP. This provides the basis for considering further work at individual plants which may allow higher log credits to be obtained on a plant by plant basis.

SPATIALAND TEMPORAL VARIATION IN CONCENTRATION OF F-SPECIFIC RNA BACTERIOPHAGES IN AN OPEN CHANNEL RECEIVING JOHKASOU EFFLUENTS

effluents, the spatial and temporal variations of the concentration of F-specific RNA bacteriophages, as model enteric viruses, were examined both in water and sediments. The investigations were carried out at six sites along an open channel in a residential area (Gifu, Japan) from September 2011 to January 2013. F-RNA bacteriophages and Qβ were detected in the open channel ranging from 0 to 10

Occurrence and reduction of human viruses, F-specific RNA coliphage genogroups and microbial indicators at a full-scale wastewater treatment plant in Japan

To evaluate and compare the reductions of human viruses and F-specific coliphages in a full-scale wastewater treatment plant based on the quantitative PCR (qPCR) and plate count assays. A total of 24 water samples were collected from four locations at the plant, and the relative abundance of human viruses and F-RNA phage genogroups were determined by qPCR. Of the 10 types of viruses tested, enteric adenoviruses were the most prevalent in both influent and effluent wastewater samples. Of the different treatment steps, the activated sludge process was most effective in reducing the microbial loads. Viruses and F-RNA phages showed variable reduction; among them, GI and GIII F-RNA phages showed the lowest and the highest reduction, respectively. Ten types of viruses were present in wastewater that is discharged into public water bodies after treatment. The variability in reduction for the different virus types demonstrates that selection of adequate viral indicators is important for evaluating the efficacy of wastewater treatment and ensuring the water safety. Our comprehensive analyses of the occurrence and reduction of viruses and indicators can contribute to the future establishment of appropriate viral indicators to evaluate the efficacy of wastewater treatment.

Concentration of Norovirus during Wastewater Treatment and Its Impact on Oyster Contamination

The concentrations of Escherichia coli, F-specific RNA bacteriophage (FRNA bacteriophage), and norovirus genogroup I (NoV GI) and norovirus genogroup II (NoV GII) in wastewater were monitored weekly over a 1-year period at a wastewater treatment plant (WWTP) providing secondary wastewater treatment. A total of 49 samples of influent wastewater and wastewater that had been treated by primary and secondary wastewater treatment processes (primary and secondary treated wastewater) were analyzed. Using a real-time reverse transcription-quantitative PCR (RT-qPCR), the mean NoV GI and NoV GII concentrations detected in effluent wastewater were 2.53 and 2.63 log(10) virus genome copies 100 ml(-1), respectively. The mean NoV concentrations in wastewater during the winter period (January to March) (n = 12) were 0.82 (NoV GI) and 1.41 (NoV GII) log units greater than the mean concentrations for the rest of the year (n = 37). The mean reductions of NoV GI and GII during treatment were 0.80 and 0.92 log units, respectively, with no significant difference detected in the extent of NoV reductions due to season. No seasonal trend was detected in the concentrations of E. coli or FRNA bacteriophage in wastewater influent and showed mean reductions of 1.49 and 2.13 log units, respectively. Mean concentrations of 3.56 and 3.72 log(10) virus genome copies 100 ml(-1) for NoV GI and GII, respectively, were detected in oysters sampled adjacent to the WWTP discharge. A strong seasonal trend was observed, and the concentrations of NoV GI and GII detected in oysters were correlated with concentrations detected in the wastewater effluent. No seasonal difference was detected in concentrations of E. coli or FRNA bacteriophage detected in oysters.

Transport of Escherichia coli and F-RNA Bacteriophages in a 5-M Column of Saturated, Heterogeneous Gravel

The relative transport and attenuation of bacteria, bacteriophages, and bromide was determined in a 5 m long × 0.3 m diameter column of saturated, heterogeneous gravel. The average pore velocity (V), longitudinal dispersivity (α x ), and total removal rate (λ) were derived from the breakthrough curves at 1, 3, and 5 m, at a flow rate of 24.8 L h−1. The experiments largely confirmed the differences in transport and attenuation patterns among bacteria, phages, and bromide, and between colloid-associated and “free” microorganisms, previously observed in a study using homogeneous pea gravel. Cultured Escherichia coli J6-2 cells were transported faster than phage MS2 and bromide, consistent with velocity enhancement of the larger particles. The evidence for velocity enhancement of phage MS2 compared with bromide was less conclusive, with some evidence of retardation of the phage as a result of adsorption–desorption processes in the finer media. On average, phage in sewage and adsorbed to kaolin particles were transported faster than free phage, suggesting that most sewage phage are adsorbed to colloids. However, average velocities of cultured and sewage E. coli differed far less, suggesting that most E. coli in sewage exist as individual (non colloid-associated) cells. There was no conclusive evidence that the wider pore size range in the heterogeneous mixture compared with pea gravel increased velocity enhancement effects. Removal rates of free phage were far higher than in the pea gravel, and were attributed to adsorption in the finer materials. Equivalent increases in removal of cultured and sewage E. coli and colloid-associated phage were attributed to straining in finer materials and settling in quiescent zones. Inactivation (μ) rates (determined in the pea gravel study) indicated little contribution to removal of either free or attached microorganisms. The results showed the importance of association with colloids in determining the relative transport of bacteria and viruses in alluvial gravels.

Transport of Escherichia coli and F-RNA bacteriophages in a 5 m column of saturated pea gravel

The relative transport and attenuation of bacteria, bacteriophages, and bromide was determined in a 5 m long × 0.3 m diameter column of saturated pea gravel. The velocity ( V), longitudinal dispersivity ( α x) and total removal rate ( λ) were calculated from the breakthrough curves at 1 m, 3 m, and 5 m, at a flow rate of 32 L h − 1 . Inactivation ( μ) rates were determined in survival chambers. Two pure culture experiments with Escherichia coli J6-2 and F-RNA phage MS2 produced an overall V ranking of E. coli J6-2 > MS2 > bromide, consistent with velocity enhancement, whereby larger particles progressively move into faster, central streamlines of saturated pores. Removal rates were near zero for MS2, but were higher for E. coli J6-2. In two sewage experiments, E. coli and F-RNA phage Vs were similar (but > bromide). This was attributed to phage adsorption to colloids similar in size to E. coli cells. Sewage phage removal rates were higher than for the pure MS2 cultures. The application of filtration theory suggested that, whereas free phage were unaffected by settling, this was the primary removal mechanism for the colloid-associated phage. However, cultured and sewage E. coli removal rates were similar, suggesting the dominance of free E. coli cells in the sewage. When MS2 was attached to kaolin particles, it was transported faster than free MS2, but at similar rates to sewage phage. The μ values indicated little contribution of inactivation to removal of either cultured or sewage microorganisms. The results showed the importance of association with colloids in determining the relative transport of bacteria and viruses in gravels.

Performance of UV disinfection and the microbial quality of greywater effluent along a reuse system for toilet flushing

This paper examines the microbial quality of treated RBC (Rotating Biological Contactor) and MBR (Membrane Bioreactor) light greywater along a continuous pilot-scale reuse system for toilet flushing, quantifies the efficiency of UV disinfection unit, and evaluates the regrowth potential of selected microorganisms along the system. The UV disinfection unit was found to be very efficient in reducing faecal coliforms and Staphylococcus aureus. On the other hand, its efficiency of inactivation of HPC (Heterotrophic Plate Count) and Pseudomonas aeruginosa was lower. Some regrowth occurred in the reuse system as a result of HPC regrowth which included opportunistic pathogens such as P. aeruginosa. Although the membrane (UF) of the MBR system removed all bacteria from the greywater, bacteria were observed in the reuse system due to “hopping phenomenon.” The microbial quality of the disinfected greywater was found to be equal or even better than the microbial quality of “clean” water in toilet bowls flushed with potable water (and used for excretion). Thus, the added health risk associated with reusing the UV-disinfected greywater for toilet flushing (regarding P. aeruginosa and S. aureus), was found to be insignificant. The UV disinfection unit totally removed (100%) the viral indicator (F-RNA phage, host: E. coli F amp +) injected to the treatment systems simulating transient viral contamination. To conclude, this work contributes to better design of UV disinfection reactors and provides an insight into the long-term behavior of selected microorganisms along on-site greywater reuse systems for toilet flushing.