The Therapeutic Potential of the “Yin-Yang” Garden in Our Gut

The gut microbiota is made up of trillion microorganisms comprising bacteria, archaea, and eukaryota living in an intimate relationship with the host. This is a highly diverse microbial community and is essentially an open ecosystem despite being deeply embedded in the human body. The gut microbiome is continually exposed to allochthonous bacteria that primarily originates from food intake. Comprising more than 1000 bacterial species, the gut microbiota endows so many different functions—so many that can be considered as an endocrine organ of its own. In this book chapter, we summarize the importance of gut microbiota in the development and maintenance of a healthy human body. We first describe how the gut microbiota is formed during the birth of a human baby and how a healthy microflora is established overtime. We also discuss how important it is to maintain the microbiota in its homeostatic condition. A discussion is also given on how alterations in the microbiota are characteristic of many diseased conditions. Recent investigations report that reestablishing a healthy microbiota in a diseased individual using fecal microbial transplant can be used as a therapeutic approach in curing many diseases. We conclude this chapter with a detailed discussion on fecal microbial transplants

Unravelling the Lipidome of Idiopathic Pulmonary Fibrosis and its Spatial Distribution using High Resolution Mass Spectrometry

Chronic lung diseases are complex, progressive disorders with increasing incidence and mortality. Chronic obstructive pulmonary disease (COPD), asthma and pulmonary fibrosis are examples of chronic lung conditions that can significantly impact the quality of life. Minimally-invasive diagnostic methods that eliminate bronchoscopic and surgical biopsy from patients are ideal; metabolomics therefore holds considerable promise for the discovery of biomarkers that can aid diagnosis and treatment with greater sensitivity, specificity and precision. The main aim of this project was to employ ultra-performance liquid chromatography-quadrupole time-of-flight (UPLC-QTOF) high resolution mass spectrometry (HRMS) and matrix-assisted laser desorption ionisation (MALDI) mass spectral imaging (MSI) together with multivariate statistics-based metabolomics to identify and characterize potential lipid biomarkers of idiopathic pulmonary fibrosis (IPF). This dissertation consists of the following studies: (1) literature review of metabolomics in chronic lung diseases; (2) application of HRMS for untargeted metabolic profiling of chronic lung disease including COPD and IPF; (3) investigation of a novel data-independent acquisition (DIA) approach to augment untargeted approaches for lipid biomarker identification; (4) development of a novel matrix application technique to improve MALDI-MSI acquisitions of tissue sections whilst maintaining spatial localisation of endogenous metabolites; and (5) exploiting potassium adduct formation to resolve the spatial distribution of lipids in fibrotic tissues. A total of 65 clinical plasma samples (from 20 healthy control subjects, 21 COPD and 24 IPF patients) were profiled using UHPLC-QTOF-MS. A fundamental challenge in using HRMS for untargeted profiling of complex, chronic lung diseases is the heterogeneity of the human samples. Various contaminations present in fibrotic tissues or adjacent non-fibrotic constituents can confound characterization and encumber the discovery of reliable biomarkers. The results of this study revealed significant correlation between COPD and IPF clinical phenotypes and plasma metabolite profiles. The unbiased metabolomics workflow and deconvolution pipeline provided end-to-end analysis from peak picking and annotation through to metabolite identification. Subsequently, the ability of the UPLC-QTOF-MS method to discriminate between lipid species was enhanced by the application of a DIA method to distinguish between “stable versus progressor” IPF patients. This DIA method is known as SONAR and uses a wide, continuously sliding precursor window for fragmentation, thereby allowing correlation of precursor and fragment ions. SONAR lipid data were processed using Progenesis QI and searched against LIPID MAPS for structural elucidation and metabolite confirmation. The lipids identified were found to be intermediates of key metabolic pathways such as the glycolytic/TCA cycle, mitochondrial-beta oxidation and lipid metabolism and hold considerable promise as biomarkers of disease. The matrix deposition step in MALDI-MSI is crucial for simultaneous extraction of metabolites from tissue sections as well as maintaining the spatial dimensionality of the endogenous metabolites. A novel, efficient and cost-effective preparative method referred to as the “freeze-spot” method was developed using wheat seed sections to demonstrate extraction efficiency and reliability, whilst maintaining the spatial resolution of the acquired MALDI-MSI images. The technique was also found to be simple and robust, forming fine matrix crystals that enabled efficient ionisation of surface metabolites, further eliminating the need for sophisticated matrix application approaches. In the final study, 10 healthy and 10 fibrotic tissues were compared using MALDI-MSI. The MSI technique developed uses potassium adduct formation to improve spatial resolution and dimensionality of lipid species such as triglycerides (TG), ceramides, sphingolipids and glycerophospholipids. The results of this study showed changes in lipid composition of IPF tissues compared to healthy controls. This study identified lysophosphatidylcholine (LysoPC), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) as potential lipid biomarkers of the disease and requires further study as targets of intervention and treatment. Both SONAR and MSI successfully identified similar classes of lipids (TG, PE, LysoPC and PC) which may play a role in the pathophysiology of the IPF lipidome. This project highlighted the complementarity of HRMS and MSI based metabolomics for the characterization of unique lipid features in fibrotic tissue and plasma samples. The study also demonstrated the discriminative power of the unbiased DIA approach for the identification of lipids via fragment ion patterns that were indicative of specific lipid classes. In addition, the application of chemometric principal component analysis (PCA) and orthogonal partial least-squares to latent structures-direct analysis (OPLS-DA) proved useful for the identification of statistically significant lipids. This statistical approach allowed for the assessment of covariance and correlation between lipids and the modelled lung diseases, and further illustrated lipid compositional changes in chronic lung diseases. Taken together, the experimental work presented in this thesis show the large potential for mass spectrometry-based metabolomics as a tool for discovery. The specificity of the novel methods outlined will be highly beneficial for compound identification and further confirmation of disease biomarkers

A Review on the Applications of Next Generation Sequencing Technologies as Applied to Food-Related Microbiome Studies

peer-reviewedThe development of next generation sequencing (NGS) techniques has enabled researchers to study and understand the world of microorganisms from broader and deeper perspectives. The contemporary advances in DNA sequencing technologies have not only enabled finer characterization of bacterial genomes but also provided deeper taxonomic identification of complex microbiomes which in its genomic essence is the combined genetic material of the microorganisms inhabiting an environment, whether the environment be a particular body econiche (e.g., human intestinal contents) or a food manufacturing facility econiche (e.g., floor drain). To date, 16S rDNA sequencing, metagenomics and metatranscriptomics are the three basic sequencing strategies used in the taxonomic identification and characterization of food-related microbiomes. These sequencing strategies have used different NGS platforms for DNA and RNA sequence identification. Traditionally, 16S rDNA sequencing has played a key role in understanding the taxonomic composition of a food-related microbiome. Recently, metagenomic approaches have resulted in improved understanding of a microbiome by providing a species-level/strain-level characterization. Further, metatranscriptomic approaches have contributed to the functional characterization of the complex interactions between different microbial communities within a single microbiome. Many studies have highlighted the use of NGS techniques in investigating the microbiome of fermented foods. However, the utilization of NGS techniques in studying the microbiome of non-fermented foods are limited. This review provides a brief overview of the advances in DNA sequencing chemistries as the technology progressed from first, next and third generations and highlights how NGS provided a deeper understanding of food-related microbiomes with special focus on non-fermented foods

Flow cytometric and 16S sequencing methodologies for monitoring the physiological status of the microbiome in powdered infant formula production

The aim of this study was to develop appropriate protocols for flow cytometric (FCM) and 16S rDNA sequencing investigation of the microbiome in a powdered infant formula (PIF) production facility. Twenty swabs were collected from each of the three care zones of a PIF production facility and used for preparing composite samples. For FCM studies, the swabs were washed in 200 mL phosphate buffer saline (PBS). The cells were harvested by three-step centrifugation followed by a single stage filtration. Cells were dispersed in fresh PBS and analyzed with a flow cytometer for membrane integrity, metabolic activity, respiratory activity and Gram characteristics of the microbiome using various fluorophores. The samples were also plated on agar plates to determine the number of culturable cells. For 16S rDNA sequencing studies, the cells were harvested by centrifugation only. Genomic DNA was extracted using a chloroform-based method and used for 16S rDNA sequencing studies. Compared to the dry low and high care zones, the wet medium care zone contained a greater number of viable, culturable, and metabolically active cells. Viable but non-culturable cells were also detected in dry-care zones. In total, 243 genera were detected in the facility of which 42 were found in all three care zones. The greatest diversity in the microbiome was observed in low care. The genera present in low, medium and high care were mostly associated with soil, water, and humans, respectively. The most prevalent genera in low, medium and high care were Pseudomonas, Acinetobacter, and Streptococcus, respectively. The integration of FCM and metagenomic data provided further information on the density of different species in the facility

Hha has a defined regulatory role that is not dependent upon H-NS or StpA

The Hha family of proteins is involved in the regulation of gene expression in enterobacteria by forming complexes with H-NS-like proteins. Whereas several amino acid residues of both proteins participate in the interaction, some of them play a key role. Residue D48 of Hha protein is essential for the interaction with H-NS, thus the D48N substitution in Hha protein abrogates H-NS/Hha interaction. Despite being a paralog of H-NS protein, StpA interacts with HhaD48N with higher affinity than with the wild type Hha protein. To analyze whether Hha is capable of acting independently of H-NS and StpA, we conducted transcriptomic analysis on the hha and stpA deletion strains and the hhaD48N substitution strain of Salmonella Typhimurium using a custom microarray. The results obtained allowed the identification of 120 genes regulated by Hha in an H-NS/StpA-independent manner, 38% of which are horizontally acquired genes. A significant number of the identified genes are involved in functions related to cell motility, iron uptake, and pathogenicity. Thus, motility assays, siderophore detection and intra-macrophage replication assays were performed to confirm the transcriptomic data. Our findings point out the importance of Hha protein as an independent regulator in S. Typhimurium, highlighting a regulatory role on virulence

RNA-seq Brings New Insights to the Intra-Macrophage Transcriptome of Salmonella Typhimurium.

Salmonella enterica serovar Typhimurium is arguably the world's best-understood bacterial pathogen. However, crucial details about the genetic programs used by the bacterium to survive and replicate in macrophages have remained obscure because of the challenge of studying gene expression of intracellular pathogens during infection. Here, we report the use of deep sequencing (RNA-seq) to reveal the transcriptional architecture and gene activity of Salmonella during infection of murine macrophages, providing new insights into the strategies used by the pathogen to survive in a bactericidal immune cell. We characterized 3583 transcriptional start sites that are active within macrophages, and highlight 11 of these as candidates for the delivery of heterologous antigens from Salmonella vaccine strains. A majority (88%) of the 280 S. Typhimurium sRNAs were expressed inside macrophages, and SPI13 and SPI2 were the most highly expressed pathogenicity islands. We identified 31 S. Typhimurium genes that were strongly up-regulated inside macrophages but expressed at very low levels during in vitro growth. The SalComMac online resource allows the visualisation of every transcript expressed during bacterial replication within mammalian cells. This primary transcriptome of intra-macrophage S.-Typhimurium describes the transcriptional start sites and the transcripts responsible for virulence traits, and catalogues the sRNAs that may play a role in the regulation of gene expression during infection

Analysis of the Oxidative Stress Regulon Identifies soxS as a Genetic Target for Resistance Reversal in Multidrug-Resistant Klebsiella pneumoniae

In bacteria, the defense system deployed to counter oxidative stress is orchestrated by three transcriptional factors, SoxS, SoxR, and OxyR. Although the regulon that these factors control is known in many bacteria, similar data are not available for Klebsiella pneumoniae. To address this data gap, oxidative stress was artificially induced in K. pneumoniae MGH78578 using paraquat and the corresponding oxidative stress regulon recorded using transcriptome sequencing (RNA-seq). The soxS gene was significantly induced during oxidative stress, and a knockout mutant was constructed to explore its functionality. The wild type and mutant were grown in the presence of paraquat and subjected to RNA-seq to elucidate the soxS regulon in K. pneumoniae MGH78578. Genes that are commonly regulated both in the oxidative stress and soxS regulons were identified and denoted as the oxidative SoxS regulon; these included a group of genes specifically regulated by SoxS. Efflux pump-encoding genes and global regulators were identified as part of this regulon. Consequently, the isogenic soxS mutant was found to exhibit a reduction in the minimum bactericidal concentration against tetracycline compared to that of the wild type. Impaired efflux activity, allowing tetracycline to be accumulated in the cytoplasm to bactericidal levels, was further evaluated using a tetraphenylphosphonium (TPP+) accumulation assay. The soxS mutant was also susceptible to tetracycline in vivo in a zebrafish embryo model. We conclude that the soxS gene could be considered a genetic target against which an inhibitor could be developed and used in combinatorial therapy to combat infections associated with multidrug-resistant K. pneumoniae. IMPORTANCE Antimicrobial resistance is a global health challenge. Few new antibiotics have been developed for use over the years, and preserving the efficacy of existing compounds is an important step to protect public health. This paper describes a study that examines the effects of exogenously induced oxidative stress on K. pneumoniae and uncovers a target that could be useful to harness as a strategy to mitigate resistance

Characterization of Cronobacter sakazakii Strains Originating from Plant-Origin Foods Using Comparative Genomic Analyses and Zebrafish Infectivity Studies

Cronobacter sakazakii continues to be isolated from ready-to-eat fresh and frozen produce, flours, dairy powders, cereals, nuts, and spices, in addition to the conventional sources of powdered infant formulae (PIF) and PIF production environments. To understand the sequence diversity, phylogenetic relationship, and virulence of C. sakazakii originating from plant-origin foods, comparative molecular and genomic analyses, and zebrafish infection (ZI) studies were applied to 88 strains. Whole genome sequences of the strains were generated for detailed bioinformatic analysis. PCR analysis showed that all strains possessed a pESA3-like virulence plasmid similar to reference C. sakazakii clinical strain BAA-894. Core genome analysis confirmed a shared genomic backbone with other C. sakazakii strains from food, clinical and environmental strains. Emerging nucleotide diversity in these plant-origin strains was highlighted using single nucleotide polymorphic alleles in 2000 core genes. DNA hybridization analyses using a pan-genomic microarray showed that these strains clustered according to sequence types (STs) identified by multi-locus sequence typing (MLST). PHASTER analysis identified 185 intact prophage gene clusters encompassing 22 different prophages, including three intact Cronobacter prophages: ENT47670, ENT39118, and phiES15. AMRFinderPlus analysis identified the CSA family class C β-lactamase gene in all strains and a plasmid-borne mcr-9.1 gene was identified in three strains. ZI studies showed that some plant-origin C. sakazakii display virulence comparable to clinical strains. Finding virulent plant-origin C. sakazakii possessing significant genomic features of clinically relevant STs suggests that these foods can serve as potential transmission vehicles and supports widening the scope of continued surveillance for this important foodborne pathogen

PPARα and PPARγ activation is associated with pleural mesothelioma invasion but therapeutic inhibition is ineffective

Mesothelioma is a cancer that typically originates in the pleura of the lungs. It rapidly invades the surrounding tissues, causing pain and shortness of breath. We compared cell lines injected either subcutaneously or intrapleurally and found that only the latter resulted in invasive and rapid growth. Pleural tumors displayed a transcriptional signature consistent with increased activity of nuclear receptors PPARα and PPARγ and with an increased abundance of endogenous PPAR-activating ligands. We found that chemical probe GW6471 is a potent, dual PPARα/γ antagonist with anti-invasive and anti-proliferative activity in vitro. However, administration of GW6471 at doses that provided sustained plasma exposure levels sufficient for inhibition of PPARα/γ transcriptional activity did not result in significant anti-mesothelioma activity in mice. Lastly, we demonstrate that the in vitro anti-tumor effect of GW6471 is off-target. We conclude that dual PPARα/γ antagonism alone is not a viable treatment modality for mesothelioma