David J. Beale

Current and Future Perspectives on the Structural Identification of Small Molecules in Biological Systems

Although significant advances have been made in recent years, the structural elucidation of small molecules continues to remain a challenging issue for metabolite profiling. Many metabolomic studies feature unknown compounds; sometimes even in the list of features identified as “statistically significant” in the study. Such metabolic “dark matter” means that much of the potential information collected by metabolomics studies is lost. Accurate structure elucidation allows researchers to identify these compounds. This in turn, facilitates downstream metabolite pathway analysis, and a better understanding of the underlying biology of the system under investigation. This review covers a range of methods for the structural elucidation of individual compounds, including those based on gas and liquid chromatography hyphenated to mass spectrometry, single and multi-dimensional nuclear magnetic resonance spectroscopy, and high-resolution mass spectrometry and includes discussion of data standardization. Future perspectives in structure elucidation are also discussed; with a focus on the potential development of instruments and techniques, in both nuclear magnetic resonance spectroscopy and mass spectrometry that, may help solve some of the current issues that are hampering the complete identification of metabolite structure and function.

Rapid identification and source-tracking of Listeria monocytogenes using MALDI-TOF mass spectrometry

Listeria monocytogenes is an important foodborne pathogen responsible for the sometimes fatal disease listeriosis. Public health concerns and stringent regulations associated with the presence of this pathogen in food and food processing environments underline the need for rapid and reliable detection and subtyping techniques. In the current study, the application of matrix assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS) as a single identification and source-tracking tool for a collection of L. monocytogenes isolates, obtained predominantly from dairy sources within Australia, was explored. The isolates were cultured on different growth media and analysed using MALDI-TOF MS at two incubation times (24 and 48 h). Whilst reliable genus-level identification was achieved from most media, identification at the species level was found to be dependent on culture conditions. Successful speciation was highest for isolates cultured on the chromogenic Agar Listeria Ottaviani Agosti agar (ALOA, 91% of isolates) and non-selective horse blood agar (HBA, 89%) for 24h. Chemometric statistical analysis of the MALDI-TOF MS data enabled source-tracking of L. monocytogenes isolates obtained from four different dairy sources. Strain-level discrimination was also observed to be influenced by culture conditions. In addition, t-test/analysis of variance (ANOVA) was used to identify potential biomarker peaks that differentiated the isolates according to their source of isolation. Source-tracking using MALDI-TOF MS was compared and correlated with the gold standard pulsed-field gel electrophoresis (PFGE) technique. The discriminatory index and the congruence between both techniques were compared using the Simpsons Diversity Index and adjusted Rand and Wallace coefficients. Overall, MALDI-TOF MS based source-tracking (using data obtained by culturing the isolates on HBA) and PFGE demonstrated good congruence with a Wallace coefficient of 0.71 and comparable discriminatory indices of 0.89 and 0.86, respectively. MALDI-TOF MS thus represents a rapid and cost-effective source-tracking technique for L. monocytogenes.

Application of metabolomics to understanding biofilms in water distribution systems: a pilot study.

Biofilms formed in pipes are known to contribute to waterborne diseases, accelerate corrosion and cause aesthetic taste and odour issues within the potable water supply network. This paper describes a pilot study, undertaken to assess the potential of using metabolomics to monitor bacterial activity in biofilms of an urban water network. Using samples from a water mains flushing programme, it was found that a profile of intracellular and extracellular metabolites associated with microbial activity could be obtained by analysing samples using gas chromatography mass spectrometry. Chemometric analysis of the chromatograms in conjunction with data from the mass spectrometer showed that it is possible to differentiate between biofilms from different pipe materials and planktonic bacteria. This research demonstrates that metabolomics has the potential for investigating biofilms and other microbial activity within water networks, and could provide a means for enhancing monitoring programmes, understanding the source of water quality complaints, and optimising water network management strategies.

A pathway to a more sustainable water sector: sustainability-based asset management

The water sectors of many countries are faced with the need to address simultaneously two overarching challenges; the need to undertake effective asset management coupled with the broader need to evolve business processes so as to embrace sustainability principles. Research has thus been undertaken into the role sustainability principles play in asset management. As part of this research, a series of 25 in-depth interviews were undertaken with water sector professionals from around Australia. Drawing on the results of these interviews, this paper outlines the conceptual relationship between asset management and sustainability along with a synthesis of the relevant opinions voiced in the interviews. The interviews indicated that the participating water authorities have made a strong commitment to sustainability, but there is a need to facilitate change processes to embed sustainability principles into business as usual practices. Interviewees also noted that asset management and sustainability are interlinked from a number of perspectives, especially in the way decision making is undertaken with respect to assets and service provision. The interviews also provided insights into the research needed to develop a holistic sustainability-based asset management framework.

Toolbox Approaches Using Molecular Markers and 16S rRNA Gene Amplicon Data Sets for Identification of Fecal Pollution in Surface Water.

ABSTRACT In this study, host-associated molecular markers and bacterial 16S rRNA gene community analysis using high-throughput sequencing were used to identify the sources of fecal pollution in environmental waters in Brisbane, Australia. A total of 92 fecal and composite wastewater samples were collected from different host groups (cat, cattle, dog, horse, human, and kangaroo), and 18 water samples were collected from six sites (BR1 to BR6) along the Brisbane River in Queensland, Australia. Bacterial communities in the fecal, wastewater, and river water samples were sequenced. Water samples were also tested for the presence of bird-associated (GFD), cattle-associated (CowM3), horse-associated, and human-associated (HF183) molecular markers, to provide multiple lines of evidence regarding the possible presence of fecal pollution associated with specific hosts. Among the 18 water samples tested, 83%, 33%, 17%, and 17% were real-time PCR positive for the GFD, HF183, CowM3, and horse markers, respectively. Among the potential sources of fecal pollution in water samples from the river, DNA sequencing tended to show relatively small contributions from wastewater treatment plants (up to 13% of sequence reads). Contributions from other animal sources were rarely detected and were very small (<3% of sequence reads). Source contributions determined via sequence analysis versus detection of molecular markers showed variable agreement. A lack of relationships among fecal indicator bacteria, host-associated molecular markers, and 16S rRNA gene community analysis data was also observed. Nonetheless, we show that bacterial community and host-associated molecular marker analyses can be combined to identify potential sources of fecal pollution in an urban river. This study is a proof of concept, and based on the results, we recommend using bacterial community analysis (where possible) along with PCR detection or quantification of host-associated molecular markers to provide information on the sources of fecal pollution in waterways.

Detection of s-triazine pesticides in natural waters by modified large-volume direct injection HPLC

There is a need for simple and inexpensive methods to quantify potentially harmful persistent pesticides often found in our water-ways and water distribution systems. This paper presents a simple, relatively inexpensive method for the detection of a group of commonly used pesticides (atrazine, simazine and hexazinone) in natural waters using large-volume direct injection high performance liquid chromatography (HPLC) utilizing a monolithic column and a single wavelength ultraviolet-visible light (UV-vis) detector. The best results for this system were obtained with a mobile phase made up of acetonitrile and water in a 30:70 ratio, a flow rate of 2.0 mL min(-1), and a detector wavelength of 230 nm. Using this method, we achieved retention times of less than three minutes, and detection limits of 5.7 microg L(-1) for atrazine, 4.7 microg L(-1) for simazine and 4.0 microg L(-1) for hexazinone. The performance of this method was validated with an inter-laboratory trial against a National Association of Testing Authorities (NATA) accredited liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method commonly used in commercial laboratories.

Sewer Performance Reporting: Factors That Influence Blockages

Managing sewer blockages represents a significant operational challenge for water utilities. In Australia, company-level blockage rates are used to compare the effectiveness of the management strategies of different utilities. Anecdotal evidence suggests that this basis may not be a fair one for comparison because blockages are influenced by a range of factors beyond management control and vary from company to company. This issue was investigated as part of a broader research effort on sewer-blockage management undertaken in conjunction with the Water Services Association of Australia (WSAA) and its members. A Web-based survey was used to collate expert opinion on factors that influence blockage rate. The identified factors were then investigated in an exploratory analysis of blockage-related data provided by two participating utilities, supported by literature reviews. The results indicate that blockage rate is influenced by a range of factors, including asset attributes, climatic conditions, water consumption, and soil type. Because these factors vary from utility to utility, this research supports the finding that company-level blockage rate in itself is not an appropriate metric for comparing management effectiveness.

The use of laboratory scale reactors to predict sensitivity to changes in operating conditions for full-scale anaerobic digestion treating municipal sewage sludge.

Anaerobic digestion of sewage sludge is highly complex and prone to inhibition, which can cause major issues for digester operators. The result is that there have been numerous investigations into changes in operational conditions, however to date all have focused on the qualitative sensitivities, neglecting the quantitative. This study therefore aimed to determine the quantitative sensitivities by using factorial design of experiments and small semi continuous reactors. Analysis showed total and volatile solids removals are chiefly influenced by retention time, with 79% and 59% of the observed results being attributed to retention time respectively, whereas biogas was mainly influenced by loading rate, 38%, and temperature, 22%. Notably the regression model fitted to the experimental data predicted full-scale performance with a high level of precision, indicating that small reactors are subject to the same sensitivity of full-scale digesters and thus can be used to predict changes loading, retention time, and temperature.

Distributions of Fecal Markers in Wastewater from Different Climatic Zones for Human Fecal Pollution Tracking in Australian Surface Waters

ABSTRACT Recreational and potable water supplies polluted with human wastewater can pose a direct health risk to humans. Therefore, sensitive detection of human fecal pollution in environmental waters is very important to water quality authorities around the globe. Microbial source tracking (MST) utilizes human fecal markers (HFMs) to detect human wastewater pollution in environmental waters. The concentrations of these markers in raw wastewater are considered important because it is likely that a marker whose concentration is high in wastewater will be more frequently detected in polluted waters. In this study, quantitative PCR (qPCR) assays were used to determine the concentrations of fecal indicator bacteria (FIB) Escherichia coli and Enterococcus spp., HFMs Bacteroides HF183, human adenoviruses (HAdVs), and polyomaviruses (HPyVs) in raw municipal wastewater influent from various climatic zones in Australia. E. coli mean concentrations in pooled human wastewater data sets (from various climatic zones) were the highest (3.2 × 106 gene copies per ml), followed by those of HF183 (8.0 × 105 gene copies per ml) and Enterococcus spp. (3.6 × 105 gene copies per ml). HAdV and HPyV concentrations were 2 to 3 orders of magnitude lower than those of FIB and HF183. Strong positive and negative correlations were observed between the FIB and HFM concentrations within and across wastewater treatment plants (WWTPs). To identify the most sensitive marker of human fecal pollution, environmental water samples were seeded with raw human wastewater. The results from the seeding experiments indicated that Bacteroides HF183 was more sensitive for detecting human fecal pollution than HAdVs and HPyVs. Since the HF183 marker can occasionally be present in nontarget animal fecal samples, it is recommended that HF183 along with a viral marker (HAdVs or HPyVs) be used for tracking human fecal pollution in Australian environmental waters.

A Review of Analytical Techniques and Their Application in Disease Diagnosis in Breathomics and Salivaomics Research

The application of metabolomics to biological samples has been a key focus in systems biology research, which is aimed at the development of rapid diagnostic methods and the creation of personalized medicine. More recently, there has been a strong focus towards this approach applied to non-invasively acquired samples, such as saliva and exhaled breath. The analysis of these biological samples, in conjunction with other sample types and traditional diagnostic tests, has resulted in faster and more reliable characterization of a range of health disorders and diseases. As the sampling process involved in collecting exhaled breath and saliva is non-intrusive as well as comparatively low-cost and uses a series of widely accepted methods, it provides researchers with easy access to the metabolites secreted by the human body. Owing to its accuracy and rapid nature, metabolomic analysis of saliva and breath (known as salivaomics and breathomics, respectively) is a rapidly growing field and has shown potential to be effective in detecting and diagnosing the early stages of numerous diseases and infections in preclinical studies. This review discusses the various collection and analyses methods currently applied in two of the least used non-invasive sample types in metabolomics, specifically their application in salivaomics and breathomics research. Some of the salient research completed in this field to date is also assessed and discussed in order to provide a basis to advocate their use and possible future scientific directions.