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

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

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

Defined Single-Gene and Multi-Gene Deletion Mutant Collections in Salmonella enterica sv Typhimurium

Artículo de publicación ISIWe constructed two collections of targeted single gene deletion (SGD) mutants and two collections of targeted multi-gene deletion (MGD) mutants in Salmonella enterica sv Typhimurium 14028s. The SGD mutant collections contain (1), 3517 mutants in which a single gene is replaced by a cassette containing a kanamycin resistance (KanR) gene oriented in the sense direction (SGD-K), and (2), 3376 mutants with a chloramphenicol resistance gene (CamR) oriented in the antisense direction (SGD-C). A combined total of 3773 individual genes were deleted across these SGD collections. The MGD collections contain mutants bearing deletions of contiguous regions of three or more genes and include (3), 198 mutants spanning 2543 genes replaced by a KanR cassette (MGD-K), and (4), 251 mutants spanning 2799 genes replaced by a CamR cassette (MGD-C). Overall, 3476 genes were deleted in at least one MGD collection. The collections with different antibiotic markers permit construction of all viable combinations of mutants in the same background. Together, the libraries allow hierarchical screening of MGDs for different phenotypic followed by screening of SGDs within the target MGD regions. The mutants of these collections are stored at BEI Resources (www.beiresources.org) and publicly available

Bistability in myo-Inositol Utilization by Salmonella enterica Serovar Typhimurium▿

The capability of Salmonella enterica serovar Typhimurium strain 14028 (S. Typhimurium 14028) to utilize myo-inositol (MI) is determined by the genomic island GEI4417/4436 carrying the iol genes that encode enzymes, transporters, and a repressor responsible for the MI catabolic pathway. In contrast to all bacteria investigated thus far, S. Typhimurium 14028 growing on MI as the sole carbon source is characterized by a remarkable long lag phase of 40 to 60 h. We report here that on solid medium with MI as the sole carbon source, this human pathogen exhibits a bistable phenotype characterized by a dissection into large colonies and a slow-growing bacterial background. This heterogeneity is reversible and therefore not caused by mutation, and it is not observed in the absence of the iol gene repressor IolR nor in the presence of at least 0.55% CO2. Bistability is correlated with the activity of the iolE promoter (PiolE), but not of PiolC or PiolD, as shown by promoter-gfp fusions. On the single-cell level, fluorescence microscopy and flow cytometry analysis revealed a gradual switch of PiolE from the “off” to the “on” status during the late lag phase and the transition to the log phase. Deletion of iolR or the addition of 0.1% NaHCO3 induced an early growth start of S. Typhimurium 14028 in minimal medium with MI. The addition of ethoxyzolamide, an inhibitor of carboanhydrases, elongated the lag phase in the presence of bicarbonate. The positive-feedback loop via repressor release and positive induction by bicarbonate-CO2 might allow S. Typhimurium 14028 to adapt to rapidly changing environments. The phenomenon described here is a novel example of bistability in substrate degradation, and, to our knowledge, is the first demonstration of gene regulation by bicarbonate-CO2 in Salmonella

Cronobacter spp.- opportunistic foodborne pathogens: an update on evolution, osmotic adaptation and pathogenesis

Purpose of Review: Cronobacter spp. are opportunistic, foodborne pathogens capable of causing severe illnesses predom- inantly in premature and low-birth-weight infants. These organisms have evolved features, which aid them to survive under harsh environmental conditions but may also contribute to pathogenesis during infection. In this review, we highlight efforts to study genetic diversity and evolutionary aspects, osmotic adaptation and pathogenesis of these pathogens. Recent Findings: Next-generation genome sequencing-based techniques elucidated a species-level bidirectional divergence driven by niche adaptation in Cronobacter spp. Whole genome comparisons and proteomics revealed genes and pathways contributing to the survival and persistence phenotype in low-moisture environments. In silico genome comparisons and appli- cation of suitable in vivo models provided answers to pathogenesis-related questions. Summary: Development and application of innovative molecular techniques and in vivo infection models have shed light on how Cronobacter spp. adapt to challenges experienced in natural, food processing and host-related environments