Application of long amplicon propidium monoazide-PCR to assess the effects of temperature and background microbiota on pathogens in river water.

The decay rates of enteric waterborne pathogens were evaluated following the introduction of Yersinia enterocolitica, Salmonella enterica, Campylobacter jejuni and Arcobacter butzleri into river water at different temperatures (5, 15 and 25°C) for a period of 28 days. To improve the accuracy of the results a molecular viability assay, long amplicon propidium monoazide-polymerase chain reaction (PMA-PCR), was used to quantify the viable cell concentration and results from PCR with and without PMA were compared. As well, the effect of background microbiota was assessed for Y. enterocolitica and S. enterica by inoculating cells into sterile and non-sterile river water. Cell persistence was improved by up to 4 log for Y. enterocolitica and 4.5 log for S. enterica in sterile river water compared to natural river water, showing that the autochthonous biological activity in river water can accelerate the die-off of introduced bacteria. Results also showed that low temperature significantly improved the persistence of all four target bacteria in non-sterile river water. There was a more rapid decline in cell concentration in samples with PMA pretreatment; therefore using PMA-PCR analysis can provide more reliable data on viable/active enteric bacteria in aquatic microcosms and allows for improved assessment of pathogens in the environment.

Influence of water quality on nitrifier regrowth in two full-scale drinking water distribution systems.

The potential for regrowth of nitrifying microorganisms was monitored in 2 full-scale chloraminated drinking water distribution systems in Ontario, Canada, over a 9-month period. Quantitative PCR was used to measure amoA genes from ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA), and these values were compared with water quality parameters that can influence nitrifier survival and growth, including total chlorine, ammonia, temperature, pH, and organic carbon. Although there were no severe nitrification episodes, AOB and AOA were frequently detected at low concentrations in samples collected from both distribution systems. A culture-based presence-absence test confirmed the presence of viable nitrifiers. AOB were usually present in similar or greater numbers than AOA in both systems. As well, AOB showed higher regrowth potential compared with AOA in both systems. Statistically significant correlations were measured between several water quality parameters of relevance to nitrification. Total chlorine was negatively correlated with both nitrifiers and heterotrophic plate count (HPC) bacteria, and ammonia levels were positively correlated with nitrifiers. Of particular importance was the strong correlation between HPC and AOB, which reinforced the usefulness of HPC as an operational parameter to measure general microbiological conditions in distribution systems.

Identifying host sources, human health risk and indicators of Cryptosporidium and Giardia in a Canadian watershed influenced by urban and rural activities.

Cryptosporidium and Giardia were characterized in a watershed in southern Ontario, Canada, over a 2½ year period. River samples were collected every two weeks, primarily near a municipal drinking water treatment plant intake. Cryptosporidium and Giardia were frequently detected with an overall occurrence rate of 88 and 97%, respectively. Giardia concentrations were higher than Cryptosporidium, with median values of 80 cysts 100 L(-1) and 12 oocysts 100 L(-1), respectively. Although pathogens rarely show a significant relationship with fecal or water quality indicators, this study determined that Cryptosporidium, but not Giardia, was significantly correlated with Escherichia coli, turbidity and river flow. There was no correlation between the two types of protozoa, and only Giardia showed a seasonal trend with higher concentrations at cold water temperatures. Cryptosporidium genotyping of all samples found that farm animals and wildlife were an important contributor of oocysts in the watershed, and that Cryptosporidium strains/genotypes of medium to high risk for human infection (C. hominis, C. parvum and C. ubiquitum) were detected in 16% of samples. This study was able to identify Cryptosporidium host sources and human health risk, and to identify differences between Cryptosporidium and Giardia occurrence in the watershed.

The detection of Yersinia enterocolitica in surface water by quantitative PCR amplification of the ail and yadA genes.

Yersinia enterocolitica has been detected in surface water, and drinking untreated water is a risk factor for infection. PCR-based methods have been used to detect Y. enterocolitica in various sample types, but quantitative studies have not been conducted in water. In this study, quantitative PCR (qPCR)-based methods targeting the Yersinia virulence genes ail and yadA were used to survey the Grand River watershed in southern Ontario, Canada. Initial testing of reference strains showed that ail and yadA PCR assays were specific for pathogenic biotypes of Y. enterocolitica; however the genes were also detected in one clinical Yersinia intermedia isolate. A survey of surface water from the Grand River watershed showed that both genes were detected at five sampling locations, with the ail and yadA genes detected in 38 and 21% of samples, respectively. Both genes were detected more frequently at colder water temperatures. A screening of Yersinia strains isolated from the watershed showed that the ail gene was detected in three Y. enterocolitica 1A/O:5 isolates. Results of this study show that Yersinia virulence genes were commonly detected in a watershed used as a source of drinking water, and that the occurrence of these genes was seasonal.

An evaluation of methods for the isolation of Yersinia enterocolitica from surface waters in the Grand River watershed.

Yersinia enterocolitica is a foodborne pathogen, but the importance of water as a route of exposure for human infection is not well known. Y. enterocolitica isolation methods were developed primarily for food and clinical samples, and may not be effective for use with environmental samples. The objective of this study was to assess the recovery of Y. enterocolitica from surface water used for drinking water treatment. Four enrichment broths and an alkaline treatment protocol were compared for the isolation of Y. enterocolitica bioserogroup 4/O:3 spiked into surface water samples. Results showed that the methods tested were not effective for the recovery of Y. enterocolitica, primarily due to inadequate inhibition of interfering background microorganisms. Using one method that showed the most potential for recovery, Yersinia spp. were isolated from rivers in southwestern Ontario, Canada, over a 17-month period. Of 200 samples analysed, Yersinia spp. were isolated from 52 samples. All river isolates belonged to non-pathogenic sub-groups, including Y. enterocolitica biotype 1A, Y. aldovae, Y. bercovieri, Y. frederiksenii, Y. intermedia, Y. kristensenii and Y. mollaretii. Results of this study show that method improvements are required to more fully understand the role of water as a source of clinically important Yersinia strains.

Development of a direct DNA extraction protocol for real-time PCR detection of Giardia lamblia from surface water.

Giardia lamblia is one of the most recognized waterborne protozoan parasites causing gastrointestinal disease. A simple but effective DNA extraction protocol for real-time PCR detection from surface water samples was developed in this study. Eleven protocols were compared, which consisted of freeze-thaw treatments (liquid N(2) and boiling water) and purification using the Qiagen DNeasy kit, together with different combinations of proteinase K, PVP360, GITC and Chelex 100 incubation. Using concentrated surface water samples spiked with G. lamblia cysts, the necessary steps for high DNA recovery were shown to be freeze-thaw, DNeasy purification and Chelex 100 incubation. Multiple rounds of freeze-thaw treatment (five cycles per round) were reported for the first time in this study to significantly increase the DNA yield from G. lamblia cysts, from ~20% after one round of freeze-thaw to 40 and 70% after two and three-rounds of freeze-thaw, respectively. More than three rounds of freeze-thaw treatment did not promote additional DNA recovery. The final protocol included three-three-rounds of freeze-thaw treatment, DNeasy purification and Chelex 100 incubation. This method was simpler, more cost-effective, and had a comparable DNA recovery to methods involving immunomagnetic separation.

Selection and evaluation of water pretreatment technologies for managed aquifer recharge (MAR) with reclaimed water.

Managed aquifer recharge with reclaimed water is a promising strategy for indirect potable reuse. However, residual contaminants in the treated wastewater effluent could potentially have adverse effects on human health. Hence, adequate water pretreatment is required. A multi-criteria approach was used to select and evaluate suitable water pretreatment technologies that can remove these critical contaminants in wastewater effluent for MAR identified in a previous study (Yuan et al., 2017). The treatment efficiency targets were calculated based on the concentrations and the suggested limits of critical contaminants. Treatment efficiency credits were then assigned to each treatment option for the removal of critical contaminants based on literature data. Treatment units that resulted in the highest efficiency credit scores were selected and combined into treatment train options, which were evaluated in terms of treatability, cost, and sustainability. This paper proposes an approach for the selection and evaluation of water treatment options, which will be helpful to guide the future implementation of MAR projects with reclaimed water.

Identification of critical contaminants in wastewater effluent for managed aquifer recharge.

Managed aquifer recharge (MAR) using highly treated effluent from municipal wastewater treatment plants has been recognized as a promising strategy for indirect potable water reuse. Treated wastewater effluent can contain a number of residual contaminants that could have adverse effects on human health, and some jurisdictions have regulations in place to govern these. For those that do not, but where reuse may be under consideration, it is of crucial importance to develop a strategy for identifying priority contaminants, which can then be used to understand the water treatment technologies that might be required. In this study, a multi-criteria approach to identify critical contaminants in wastewater effluent for MAR was developed and applied using a case study site located in southern Ontario, Canada. An important aspect of this approach was the selection of representative compounds for each group of contaminants, based on potential for occurrence in wastewater and expected health or environmental impacts. Due to a lack of MAR regulations in Canada, the study first proposed potential recharge water quality targets. Predominant contaminants, potential additional contaminants, and potential emerging contaminants, which together comprise critical contaminants for MAR with reclaimed water, were then selected based on the case study wastewater effluent monitoring data and literature data. This paper proposes an approach for critical contaminant selection, which will be helpful to guide future implementation of MAR projects using wastewater treatment plant effluents.

Application of flow cytometry to monitor assimilable organic carbon (AOC) and microbial community changes in water.

Flow cytometry is an efficient monitoring tool for rapid cell counting, and can be applied to research on water quality and treatment. In this study, a method that employs flow cytometry and a natural microbial inoculum to determine assimilable organic carbon (AOC) was adapted for use with challenging surface waters that have a high organic and particle content, and subsequently applied in a long term river water study. AOC method optimization showed that river water bacteria could pass through a 0.2µm membrane filter, and therefore membrane filtration combined with heat treatment was required for sample sterilization. Preparation of the natural river inoculum with an acceptable yield value could only be achieved when grown using the natural water source, since growth was limited on different types of inorganic minimal media and in natural spring water. The resulting flow cytometry AOC method was reliable and reproducible, and results were comparable to the standard plate count AOC method. Size exclusion chromatography showed that both high and low molecular weight organic matter fractions were utilized by the natural AOC inoculum. Flow cytometry was used to measure both AOC levels and total cell counts in a long term study to monitor the water quality of a river which was used as a drinking water source. The method could distinguish between high nucleic acid (HNA) and low nucleic acid (LNA) groups of bacteria, and HNA bacteria were found to respond faster than LNA bacteria to seasonal changes in nutrients and water temperature.

Kinetics of natural organic matter (NOM) removal during drinking water biofiltration using different NOM characterization approaches.

To better understand biofiltration, concentration profiles of various natural organic matter (NOM) components throughout a pilot-scale drinking water biofilter were investigated using liquid chromatography - organic carbon detection (LC-OCD) and fluorescence excitation and emission matrices (FEEM). Over a 2 month period, water samples were collected from six ports at different biofilter media depths. Results showed substantial removal of biopolymers (i.e. high molecular weight (MW) NOM components as characterized by LC-OCD) and FEEM protein-like materials, but low removal of humic substances, building blocks and low MW neutrals and low MW acids. For the first time, relative biodegradability of different NOM components characterized by LC-OCD and FEEM approaches were investigated across the entire MW range and for different fluorophore compositions, in addition to establishing the biodegradation kinetics. The removal kinetics for FEEM protein-like materials were different than for the LC-OCD-based biopolymers, illustrating the complementary nature of the LC-OCD and FEEM approaches. LC-OCD biopolymers (both organic carbon and organic nitrogen) and FEEM protein-like materials were shown to follow either first or second order biodegradation kinetics. Due to the low percent removal and small number of data points, the performance of three kinetic models was not distinguishable for humic substances. Pre-filtration of samples for FEEM analyses affected the removal behaviours and/or kinetics especially of protein-like materials which was attributed to the removal of the colloidal/particulate materials.

Effect of hydraulically reversible and hydraulically irreversible fouling on the removal of MS2 and φX174 bacteriophage by an ultrafiltration membrane.

The effect of membrane fouling on the removal of enteric virus surrogates MS2 and φX174 bacteriophage by an ultrafiltration membrane was assessed under simulated full-scale drinking water treatment operating conditions. Filtration experiments of up to 8 days using either river or lake water ascertained how the membrane fouling layer affected virus removal. Organic carbon fractionation techniques identified potential foulants, including biopolymers, in the feed water and in the permeate. Hydraulically irreversible fouling could greatly improve the removal of both viruses at moderate and severe fouling conditions by up to 2.5 logs. Hydraulically reversible fouling increased virus removal only slightly, and increased removal of >0.5 log for both phage were only obtained under severe fouling conditions. The increase in virus removal due to irreversible and reversible fouling differed between the two water sources. As the degree of fouling increased, differences between the removal of the two phage decreased. Maintenance cleaning partially removed membrane foulants, however virus removal following maintenance cleaning was lower than that of the fouled membrane, it remained higher than that of the clean membrane.

Size-dependent PCR inhibitory effect induced by gold nanoparticles.

In this work, the effect of gold nanoparticles (AuNPs) with diameters of around 5, 10 and 20 nm on PCR efficiency is evaluated respectively using a real-time PCR machine. Gold nanoparticles show no obvious effect on PCR at low particle concentration. When the concentration is increased, PCR inhibition is observed. At the same particle concentration, gold nanoparticles of different sizes show different inhibitory effects on PCR. It is found that Taq polymerase can interact with AuNPs. The interaction is probably due to the binding of polymerase to AuNPs therefore lowering the concentration of free polymerase. It is also found that bovine serum albumin can interact with gold nanoparticles. It is believed that BSA blocks the surface of AuNPs from forming biding sites for polymerase. It can be used as an additive to reverse the inhibitory effect caused by gold nanoparticles.

Molecular biological detection of anaerobic gut fungi (Neocallimastigales) from landfill sites.

Oligonucleotide primers were designed for the 18S rRNA genes of members of the Neocallimastigales and used in a nested PCR protocol to amplify 787-bp fragments of DNA from landfill site samples. The specificities of the primers were confirmed by phylogenetic analysis of the environmental clone sequences, and this method can therefore now be used to investigate the ecology of the obligately anaerobic fungi. To our knowledge, this is the first demonstration of the occurrence of members of the Neocallimastigales outside the mammalian gut, and their distribution across the landfill samples examined here suggests that they are actively involved in cellulose degradation.