Michele I. Van Dyke

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.

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.

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 N2 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.

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.

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.

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.

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.

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.

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.

Development and application of an improved protocol to characterize biofilms in biologically active drinking water filters

Biofilms are central to the functioning of biologically active filters for drinking water production, and accurate biofilm characterization is crucial for elucidating biofiltration mechanisms and for identifying parameters indicative of biofilter performance. Drawing on recent findings in environmental engineering and microbiology, the objective was to develop and apply an improved, comprehensive biofilm characterization protocol to accurately measure the cellular and extracellular polymeric substance (EPS) composition in biofilms attached to drinking water biofilters. Key is the cation exchange resin extraction procedure, which was adapted and optimized to separate biofilm cells and EPS directly from the biofilter media while minimizing cell lysis. This step simplifies the overall procedure by combining biofilm detachment with biomass and EPS extraction steps and has not yet been applied to drinking water biofilters. Biomass was measured using both ATP and flow cytometry, and EPS composition was determined using spectrophotometric methods for total proteins and carbohydrates, and more advanced analytical techniques including fluorescence spectroscopy and liquid chromatography with organic carbon detection. This protocol was applied to pilot-scale biofilters, and the unique combination of characterization techniques allowed relationships among parameters to be identified. There were strong correlations between the cellular and EPS components and cellular ATP content, which had not been shown previously for drinking water biofilters. The EPS protein to carbohydrate ratio and the EPS C/N ratio were stable over the monitoring period. As well, nearly 50% of the biofilm EPS had a molecular weight larger than 2000 Da, composed of predominantly protein-like substances.