Avinash V. Karpe

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.

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.

Fecal microbiota and metabolome in a mouse model of spontaneous chronic colitis: Relevance to human inflammatory bowel disease

Background:Dysbiosis of the gut microbiota may be involved in the pathogenesis of inflammatory bowel disease (IBD). However, the mechanisms underlying the role of the intestinal microbiome and metabolome in IBD onset and its alteration during active treatment and recovery remain unknown. Animal models of chronic intestinal inflammation with similar microbial and metabolomic profiles would enable investigation of these mechanisms and development of more effective treatments. Recently, the Winnie mouse model of colitis closely representing the clinical symptoms and characteristics of human IBD has been developed. In this study, we have analyzed fecal microbial and metabolomic profiles in Winnie mice and discussed their relevance to human IBD. Methods:The 16S rRNA gene was sequenced from fecal DNA of Winnie and C57BL/6 mice to define operational taxonomic units at ≥97% similarity threshold. Metabolomic profiling of the same fecal samples was performed by gas chromatography–mass spectrometry. Results:Composition of the dominant microbiota was disturbed, and prominent differences were evident at all levels of the intestinal microbiome in fecal samples from Winnie mice, similar to observations in patients with IBD. Metabolomic profiling revealed that chronic colitis in Winnie mice upregulated production of metabolites and altered several metabolic pathways, mostly affecting amino acid synthesis and breakdown of monosaccharides to short chain fatty acids. Conclusions:Significant dysbiosis in the Winnie mouse gut replicates many changes observed in patients with IBD. These results provide justification for the suitability of this model to investigate mechanisms underlying the role of intestinal microbiota and metabolome in the pathophysiology of IBD.

Untargeted metabolic profiling of Vitis Vinifera during fungal degradation

This paper illustrates the application of an untargeted metabolic profiling analysis of winery-derived biomass degraded using four filamentous fungi (Trichoderma harzianum, Aspergillus niger, Penicillium chrysogenum and P. citrinum) and a yeast (Saccharomyces cerevisiae). Analysis of the metabolome resulted in the identification of 233 significant peak features [P < 0.05; fold change (FC) > 2 and signal-to-noise ratio >50] using gas chromatography-mass spectrometry followed by statistical chemometric analysis. Furthermore, A. niger and P. chrysogenum produced higher biomass degradation due to considerable β-glucosidase and xylanase activities. The major metabolites generated during fungal degradation which differentiated the metabolic profiles of fungi included sugars, sugar acids, organic acids and fatty acids. Although, P. chrysogenum could degrade hemicelluloses due to its high β-glucosidase and xylanase activities, it could not utilize the resultant pentoses, which A. niger and P. citrinum could do efficiently, thus indicating a need of mixed fungal culture to improve the biomass degradation. Saccharomyces cerevisiae, a non-cellulose degrader, exhibited sugar accumulation during the fermentation. Penicillium chrysogenum was observed to degrade about 2% lignin, a property not observed in other fungi. This study emphasized the differential fungal metabolic behavior and demonstrated the potential of metabolomics in optimizing degradation or manipulating pathways to increase yields of products of interest.

A Community Multi-Omics Approach towards the Assessment of Surface Water Quality in an Urban River System

A multi-omics approach was applied to an urban river system (the Brisbane River (BR), Queensland, Australia) in order to investigate surface water quality and characterize the bacterial population with respect to water contaminants. To do this, bacterial metagenomic amplicon-sequencing using Illumina next-generation sequencing (NGS) of the V5–V6 hypervariable regions of the 16S rRNA gene and untargeted community metabolomics using gas chromatography coupled with mass spectrometry (GC-MS) were utilized. The multi-omics data, in combination with fecal indicator bacteria (FIB) counts, trace metal concentrations (by inductively coupled plasma mass spectrometry (ICP-MS)) and in-situ water quality measurements collected from various locations along the BR were then used to assess the health of the river ecosystem. Sites sampled represented the transition from less affected (upstream) to polluted (downstream) environments along the BR. Chemometric analysis of the combined datasets indicated a clear separation between the sampled environments. Burkholderiales and Cyanobacteria were common key factors for differentiation of pristine waters. Increased sugar alcohol and short-chain fatty acid production was observed by Actinomycetales and Rhodospirillaceae that are known to form biofilms in urban polluted and brackish waters. Results from this study indicate that a multi-omics approach enables a deep understanding of the health of an aquatic ecosystem, providing insight into the bacterial diversity present and the metabolic output of the population when exposed to environmental contaminants.

Untargeted metabolic profiling of winery-derived biomass waste degradation by Penicillium chrysogenum.

Winery-derived biomass waste was degraded by Penicillium chrysogenum under solid state fermentation over 8 days in a (2)H2O-supplemented medium. Multivariate statistical analysis of the gas chromatography-mass spectrometry (GC-MS) data resulted in the identification of 94 significant metabolites, within 28 different metabolic pathways. The majority of biomass sugars were utilized by day 4 to yield products such as sugars, fatty acids, isoprenoids, and amino acids. The fungus was observed to metabolize xylose to xylitol, an intermediate of ethanol production. However, enzyme inhibition and autolysis were observed from day 6, indicating 5 days as the optimal time for fermentation. P. chrysogenum displayed metabolism of pentoses (to alcohols) and degraded tannins and lignins, properties that are lacking in other biomass-degrading ascomycetes. Rapid fermentation (3-5 days) may not only increase the pentose metabolizing efficiency but also increase the yield of medicinally important metabolites, such as syringate.

Metabolic profiling and in vitro assessment of anthelmintic fractions of Picria fel-terrae Lour.

Anthelmintic resistance is widespread in gastrointestinal nematode populations, such that there is a consistent need to search for new anthelmintics. However, the cost of screening for new compounds is high and has a very low success rate. Using the knowledge of traditional healers from Borneo Rainforests (Sarawak, Malaysia), we have previously shown that some traditional medicinal plants are a rich source of potential new anthelmintic drug candidates. In this study, Picria fel-terrae Lour. plant extract, which has previously shown promising anthelmintic activities, was fractionated via the use of a solid phase extraction cartridge and each isolated fraction was then tested on free-living nematode Caenorhabditis elegans and the parasitic nematode Haemonchus contortus. We found that a single fraction was enriched for nematocidal activity, killing ≥90% of C. elegans adults and inhibiting the motility of exsheathed L3 of H. contortus, while having minimal cytotoxic activity in mammalian cell culture. Metabolic profiling and chemometric analysis of the effective fraction indicated medium chained fatty acids and phenolic acids were highly represented.

Beyond Metabolomics: A Review of Multi-Omics-Based Approaches

Genomics and transcriptonomics studies have been the cornerstones of numerous biological progresses in several decades. Additionally, proteomic studies, especially related to clinical studies have been popular since 1970s. In last decade, owing to technological advances, considerable progress has been made in the metabolic studies of several biological phenomena, ranging from clinical to environmental studies. Since the advent of the Human Genome consortium in the 1990s, there has been an exponential progress in the studies of not only genomics (and metagenomics), but also transcriptomics and proteomics. A rapid rise in metabolomics and its complementation to the former researches has necessitated the combination of all these techniques to obtain a broader picture of the biological system. A combination study, broadly termed as ‘Multi-omics’ has been a field of interest since 2008. A ‘multi-omics’ approach combines major fields such as genomics, transcriptomics, proteomics and metabolomics, in addition to minor omics-based approaches such as fluxomics, ionomics and lipidomics (among others) in order to obtain elaborate knowledge of an entire biological system, in a single study. This chapter discusses the progress of multi-omics in recent years and presents some case studies in multiple fields such as human health, the environment, industrial biotechnology and biofuel studies among others. The chapter also discusses the development of various analytical methods and databases in multi-omics, with tentative future trends in the field.

Omics-based approaches and their use in the assessment of microbial-influenced corrosion of metals

Abstract Microbial-influenced corrosion (MIC) has been known to have economic, environmental, and social implications to offshore oil and gas pipelines, concrete structures, and piped water assets. While corrosion itself is a relatively simple process, the localised manner of corrosion makes in situ assessments difficult. Furthermore, corrosion assessments tend to be measured as part of a forensic investigation. Compounding the issue further is the impact of microbiological/biofilm processes, where corrosion is influenced by the complex processes of different microorganisms performing different electrochemical reactions and secreting proteins and metabolites that can have secondary effects. While traditional microbiological culture-dependent techniques and electrochemical/physical assessments provide some insight into corrosion activity, the identity and role of microbial communities that are related to corrosion and corrosion inhibition in different materials and in different environments are scarce. One avenue to explore MIC and MIC inhibition is through the application of omics-based techniques, where insight into the bacterial population in terms of diversification and their metabolism can be further understood. As such, this paper discusses the recent progresses made in a number of fields that have used omics-based applications to improve the fundamental understanding of biofilms and MIC processes.

A multi-omics based ecological analysis of coastal marine sediments from Gladstone, in Australia’s Central Queensland, and Heron Island, a nearby fringing platform reef

The impact of anthropogenic factors arising from point and non-point pollution sources at a multi commodity marine port and its surrounding ecosystems were studied using sediment samples collected from a number of onshore (Gladstone Harbour and Facing Island) and offshore (Heron Island and Fitzroy Reefs) sites in Australias Central Queensland. Sediment samples were analyzed for trace metals, organic carbon, polycyclic aromatic hydrocarbons (PAH), emerging chemicals of concern (ECC) and sterols. Similarly, the biological and biochemical interaction between the reef and its environment was analyzed by the multi-omic tools of next-generation sequencing characterization of the bacterial community and microbial community metabolic profiling. Overall, the trace elements were observed at the lower end of the Australian environmental guideline values at the offshore sites, while higher values were observed for the onshore locations Nickel and copper were observed above the high trigger value threshold at the onshore sites. The levels of PAH were below limits of detection across all sites. However, some of the ECC and sterols were observed at higher concentrations at both onshore and offshore locations, notably, the cholesterol family sterols and 17α-ethynylestradiol. Multi-omic analyses also indicated possible thermal and photo irradiation stressors on the bacterial communities at all the tested sites. The observed populations of γ-proteobacteria were found in combination with an increased pool of fatty acids that indicate fatty acid synthesis and utilisation of the intermediates of the shikimate pathways. This study demonstrates the value of applying a multi-omics approach for ecological assessments, in which a more detailed assessment of physical and chemical contaminants and their impact on the community bacterial biome is obtained.