Kimberley A. Gilbride

Evaluation of low-copy genetic targets for waterborne bacterial pathogen detection via qPCR.

Recent developments in water quality research have highlighted difficulties in accurately predicting the incidence of pathogens within freshwater based on the viability, culturability and metabolic activity of indicator organisms. QPCR-driven assays are candidates to replace standard culture-based methods, however, protocols suitable for routine use have yet to be sufficiently validated. The objective of this study was to evaluate five oligonucleotide primers sets (ETIR, SINV, exoT, VS1 and ipaH2) for their potential applicability in qPCR assays to detect contamination from five waterborne bacterial pathogens (Escherichia coli O157:H7, Salmonella Typhimurium, Campylobacter jejuni, Pseudomonas aeruginosa, and Shigella flexneri). An enrichment-free qPCR protocol was also tested using S. Typhimurium-seeded source water, combining membrane filtration and mechanical, chemical and enzymatic lysis techniques to recover the bacterial cells. All five primer sets were found to have high specificity and sensitivity for the tested organisms. Four of the primers were able to detect pathogen loads as low as 10 cells/mL while 200 cells/mL of C. jejuni were detectable in pure culture. Although sensitivity decreased in an artificially contaminated environmental matrix, it was still possible to detect as few as 10 S. Typhimurium cells without enrichment. The primers and protocols evaluated in this study have demonstrated potential for further validation for possible application alongside traditional indicator techniques.

Impact of protozoan grazing on nitrification and the ammonia- and nitrite-oxidizing bacterial communities in activated sludge

In activated sludge, protozoa feed on free-swimming bacteria and suspended particles, inducing flocculation and increasing the turnover rate of nutrients. In this study, the effect of protozoan grazing on nitrification rates under various conditions in municipal activated sludge batch reactors was examined, as was the spatial distribution of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) within the activated sludge. The reactors were monitored for ammonia, nitrite, nitrate, and total nitrogen concentrations, and bacterial numbers in the presence and absence of cycloheximide (a protozoan inhibitor), allylthiourea (an inhibitor of ammonia oxidation), and EDTA (a deflocculating agent). The accumulations of nitrate, nitrite, and ammonia were lower in batches without than with protozoa grazing. Inhibition of ammonia oxidation also decreased the amount of nitrite and nitrate accumulation. Inhibiting protozoan grazing along with ammonia oxidation further decreased the amounts of nitrite and nitrate accumulated. Induction of deflocculation led to high nitrate accumulation, indicating high levels of nitrification; this effect was lessened in the absence of protozoan grazing. Using fluorescent in situ hybridization and confocal laser scanning microscopy, AOB and NOB were found clustered within the floc, and inhibiting the protozoa, inhibiting ammonia oxidation, or inducing flocculation did not appear to lower the number of AOB and NOB present or affect their position within the floc. These results suggest that the AOB and NOB are present but less active in the absence of protozoa.

Detection of melamine and cyanuric acid in vegetable protein products used in food production.

The multitude of food recalls in 2007 clearly demonstrated that total nitrogen-content (ΣN) determination by means of near-infrared spectroscopy (NIRS) and Kjeldahl-based measurements can be deceived, and should no longer be regarded as a complete quality assurance program for nutritive-protein evaluations. Furthermore, contemporary Canadian-employed analytical tools are precariously limited in their ability to effectively assure a product where there is no a priori knowledge of the environmental toxin(s) involved. In light of these challenges, this study explored a number of analytical techniques used to assess and furthermore assure the quality of vegetable protein products (VPPs). Using liquid chromatography with tandem mass spectrometry (LC/MS/MS) technologies, a combination of VPP-based samples was analyzed for the presence of nitrogen-bearing environmental toxicants. Of the 52 samples tested, involving an assortment of matrices, melamine and cyanuric acid were positively identified (>1 ng/mL) in 22 and 17 samples, respectively. Subsequent high pressure liquid chromatography with ultraviolet/visible (HPLC-UV) amino acid profiling further confirmed the adulteration of those materials contaminated with melamine and melamine-related compounds. Based on the evidence presented herein, LC/MS/MS in combination with HPLC-UV provides for a reliable food safety detection system as applied to VPPs. Moreover, HPLC-UV is indispensable as a stand-alone 1st level of screening to assess the integrity of a VPP or any nutritive protein-based sample.

Molecular Methods for the Detection of Waterborne Pathogens

Abstract The safety of potable water relies on sensitive and specific testing of samples for pathogenic waterborne agents. Traditional methods often fail to recover or detect the causative pathogen and rely on the detection of indicator organisms to judge the safety of the samples. Molecular technology has grown enormously in the past several decades and lends itself to the development of rapid, sensitive and specific tests for pathogens in all types of samples. This chapter reviews the molecular methods that have been designed and used to detect viruses, bacteria, protozoa, and cyanobacteria from water and environmental samples. It gives a brief description of each method followed by examples of where the method has been used. Detection limits of each method are noted where available along with advantages and disadvantages of each technique.

Utilization of multiple organisms in a proposed early-warning biomonitoring system for real-time detection of contaminants: preliminary results and modeling

During past decades, biomonitors were deployed in lakes and rivers to rapidly detect hazardous chemicals by measuring the endpoints of a single aquatic species at defined short intervals. Most biomonitors, however, are only capable of indicating a departure from baseline water conditions without identifying the cause. In order to provide a more comprehensive assessment, a biomonitoring system which features a library of stereotyped responses of multiple aquatic species in various water conditions is proposed. A preliminary library was constructed by characterizing the behavioural and physiological responses of Daphnia magna, Hyalella azteca, Lumbriculus variegatus, and Pseudokirchneriella subcapitata to various concentrations of atrazine and tributyltin. By employing multivariate statistical tools such as principal component analysis (PCA) and discriminant analysis, this library (which contained responses after 6h of exposure to contaminants) was used as a template to classify and to model other sets of earlier measurements at 2 and 4h, resulting in an accuracy of 73 and 97%, respectively. These findings demonstrated the potential capability of the proposed early-warning biomonitoring system to provide real-time water quality assessment and early-warning contaminant detection.

Effects of tetracycline and ibuprofen on the relative abundance of microbial eukaryotic and bacterial populations in wastewater treatment

The activated sludge process in a wastewater treatment plant (WWTP) relies on the activity of microbes to reduce the organic and inorganic matter and produce effluent that is safe to discharge into receiving waters. This research examined the effects of the non-steroidal anti-inflammatory drug ibuprofen and the antibiotic tetracycline on the relative abundance and composition of eukaryotes and bacteria in the microbial population present in activated sludge from a WWTP. The current investigation was designed to observe the impact of these contaminants, at low (environmentally relevant concentrations) as well as high concentrations of the drugs. Using 16S and 18S rRNA gene primer sets and quantitative polymerase chain reaction, the abundance of each population was monitored as well as the relative ratio of the two populations under the various conditions. It was found that current environmentally relevant concentrations of ibuprofen (100 ng/mL) stimulated eukaryotic growth but higher concentrations (2,000 ng/mL, 100,000 ng/mL) reduced their numbers significantly especially in the presence of tetracycline. Finally using denaturing gradient gel electrophoresis, some of the more abundant eukaryotes were identified and it was noted that high ibuprofen and tetracycline concentrations favoured the abundance of some genera.

A closer look at the antibiotic-resistant bacterial community found in urban wastewater treatment systems

The conventional biological treatment process can provide a favorable environment for the maintenance and dissemination of antibiotic‐resistant bacteria and the antibiotic resistance genes (ARG) they carry. This study investigated the occurrence of antibiotic resistance in three wastewater treatment plants (WWTP) to determine the role they play in the dissemination of ARGs. Bacterial isolates resistant to tetracycline were collected, and tested against eight antibiotics to determine their resistance profiles and the prevalence of multiple antibiotic resistance. It was found that bacteria resistant to tetracycline were more likely to display resistance to multiple antibiotics compared to those isolates that were not tetracycline resistant. Polymerase chain reaction (PCR) was used to identify the tetracycline resistance determinants present within the bacterial communities of the WWTPs and receiving waters, and it was found that ARGs may not be released from the treatment process. Identification of isolates showed that there was a large diversity of species in both the tetracycline‐resistant and tetracycline‐sensitive populations and that the two groups were significantly different in composition. Antibiotic resistance profiles of each population showed that a large diversity of resistance patterns existed within genera suggesting that transmission of ARG may progress by both horizontal gene and vertical proliferation.

Automated image analysis of Euglena gracilis Klebs (Euglenophyta) for measuring sublethal effects of three model contaminants.

The short-term impacts of atrazine (herbicide), tributyltin (organometal) and copper on the behaviour of Euglena gracilis Klebs (Euglenophyta) were assessed. First, the ECOTOX automated image analysis system was used, which measured swimming velocity, cell shape, percentage of cells swimming upwards, and randomness of swimming. Next, visual observation by microscopy was used to measure percentage of cell motility and cell shape. Behavioural changes can be used as an indicator of stress in less than 24 h, potentially making them suitable for inclusion in early-warning systems for water quality. Findings indicate that E. gracilis is a very sensitive organism to copper, showing inhibition of motility with visual observation at 0.8 μmol/L within 1 h. The image analysis system was in general less sensitive than visual observation for detecting behavioural changes after incubation in copper. In contrast, after exposure to organic contaminants atrazine and tributyltin, the ECOTOX system detected small changes in the number of cells swimming upwards (antigravitactic behaviour) at higher concentrations.