Spatial chemistry of citrus reveals molecules bactericidal to Candidatus Liberibacter asiaticus.

Huanglongbing (HLB), associated with the psyllid-vectored phloem-limited bacterium, Candidatus Liberibacter asiaticus (CLas), is a disease threat to all citrus production worldwide. Currently, there are no sustainable curative or prophylactic treatments available. In this study, we utilized mass spectrometry (MS)-based metabolomics in combination with 3D molecular mapping to visualize complex chemistries within plant tissues to explore how these chemistries change in vivo in HLB-infected trees. We demonstrate how spatial information from molecular maps of branches and single leaves yields insight into the biology not accessible otherwise. In particular, we found evidence that flavonoid biosynthesis is disrupted in HLB-infected trees, and an increase in the polyamine, feruloylputrescine, is highly correlated with an increase in disease severity. Based on mechanistic details revealed by these molecular maps, followed by metabolic modeling, we formulated and tested the hypothesis that CLas infection either directly or indirectly converts the precursor compound, ferulic acid, to feruloylputrescine to suppress the antimicrobial effects of ferulic acid and biosynthetically downstream flavonoids. Using in vitro bioassays, we demonstrated that ferulic acid and bioflavonoids are indeed highly bactericidal to CLas, with the activity on par with a reference antibiotic, oxytetracycline, recently approved for HLB management. We propose these compounds should be evaluated as therapeutics alternatives to the antibiotics for HLB treatment. Overall, the utilized 3D metabolic mapping approach provides a promising methodological framework to identify pathogen-specific inhibitory compounds in planta for potential prophylactic or therapeutic applications.

Ordering molecular diversity in untargeted metabolomics via molecular community networking.

Nature's molecular diversity is not random but displays intricate organization stemming from biological necessity. Molecular networking connects metabolites with structural similarity, enabling molecular discoveries from mass spectrometry data using arbitrary similarity thresholds that can fracture natural metabolite families. We present molecular community networking (MCN), that optimizes connectivity for each metabolite, rescuing lost relationships and capturing otherwise "hidden" metabolite connections. Using MCN, we demonstrate the discovery of novel dipeptide-conjugated bile acids.

Open access repository-scale propagated nearest neighbor suspect spectral library for untargeted metabolomics.

Despite the increasing availability of tandem mass spectrometry (MS/MS) community spectral libraries for untargeted metabolomics over the past decade, the majority of acquired MS/MS spectra remain uninterpreted. To further aid in interpreting unannotated spectra, we created a nearest neighbor suspect spectral library, consisting of 87,916 annotated MS/MS spectra derived from hundreds of millions of MS/MS spectra originating from published untargeted metabolomics experiments. Entries in this library, or "suspects," were derived from unannotated spectra that could be linked in a molecular network to an annotated spectrum. Annotations were propagated to unknowns based on structural relationships to reference molecules using MS/MS-based spectrum alignment. We demonstrate the broad relevance of the nearest neighbor suspect spectral library through representative examples of propagation-based annotation of acylcarnitines, bacterial and plant natural products, and drug metabolism. Our results also highlight how the library can help to better understand an Alzheimer's brain phenotype. The nearest neighbor suspect spectral library is openly available for download or for data analysis through the GNPS platform to help investigators hypothesize candidate structures for unknown MS/MS spectra in untargeted metabolomics data.

Sniffing out safety: canine detection and identification of SARS-CoV-2 infection from armpit sweat.

Detection dogs were trained to detect SARS-CoV-2 infection based on armpit sweat odor. Sweat samples were collected using cotton pads under the armpits of negative and positive human patients, confirmed by qPCR, for periods of 15-30 min. Multiple hospitals and organizations throughout Belgium participated in this study. The sweat samples were stored at -20°C prior to being used for training purposes. Six dogs were trained under controlled atmosphere conditions for 2-3 months. After training, a 7-day validation period was conducted to assess the dogs' performances. The detection dogs exhibited an overall sensitivity of 81%, specificity of 98%, and an accuracy of 95%. After validation, training continued for 3 months, during which the dogs' performances remained the same. Gas chromatography/mass spectrometry (GC/MS) analysis revealed a unique sweat scent associated with SARS-CoV-2 positive sweat samples. This scent consisted of a wide variety of volatiles, including breakdown compounds of antiviral fatty acids, skin proteins and neurotransmitters/hormones. An acceptability survey conducted in Belgium demonstrated an overall high acceptability and enthusiasm toward the use of detection dogs for SARS-CoV-2 detection. Compared to qPCR and previous canine studies, the detection dogs have good performances in detecting SARS-CoV-2 infection in humans, using frozen sweat samples from the armpits. As a result, they can be used as an accurate pre-screening tool in various field settings alongside the PCR test.

Multi-omic approach to decipher the impact of skincare products with pre/postbiotics on skin microbiome and metabolome.

Introduction: Although pre/pro/postbiotics have become more prevalent in dermatologic and cosmetic fields, the mode of action when topically applied is largely unknown. A multi-omic approach was applied to decipher the impact of the skincare products with pre/postbiotics on skin microbiome and metabolome. Methods: Subjects with dry skin applied a body wash and body lotion with or without pre/postbiotics for 6 weeks. Skin hydration was measured at baseline, 3 and 6 weeks. Skin swabs were collected for 16S rRNA gene sequencing, metagenomics and metabolomics analysis. Results: Skin hydration significantly increased in both groups. The prebiotic group significantly reduced opportunistic pathogens, e.g., Pseudomonas stutzeri and Sphingomonas anadarae, and increased the commensals, e.g., Staphylococcus equorum, Streptococcus mitis, Halomonas desiderata. Bacterial sugar degradation pathways were enriched in the prebiotic group, while fatty acid biosynthesis pathways were reduced in control. The changes on skin metabolome profiles by the products were more prominent. The prebiotic group performed greater modulation on many clinically-relevant metabolites compared to control. Correlation analysis showed H. desiderata and S. mitis positively correlated with skin hydration, P. stutzeri and S. anadarae negatively correlated with the metabolites that are positively associated with skin hydration improvement. Conclusion: This holistic study supported a hypothesis that the pre/postbiotics increased skin hydration through the modulation of skin microbiome, metabolic pathways and metabolome.

The Molecular Effect of Wearing Silver-Threaded Clothing on the Human Skin.

With growing awareness that what we put in and on our bodies affects our health and wellbeing, little is still known about the impact of textiles on the human skin. Athletic wear often uses silver threading to improve hygiene, but little is known about its effect on the body's largest organ. In this study, we investigated the impact of such clothing on the skin's chemistry and microbiome. Samples were collected from different body sites of a dozen volunteers over the course of 12 weeks. The changes induced by the antibacterial clothing were specific for individuals, but more so defined by gender and body site. Unexpectedly, the microbial biomass on skin increased in the majority of the volunteers when wearing silver-threaded T-shirts. Although the most abundant taxa remained unaffected, silver caused an increase in diversity and richness of low-abundant bacteria and a decrease in chemical diversity. Both effects were mainly observed for women. The hallmark of the induced changes was an increase in the abundance of various monounsaturated fatty acids (MUFAs), especially in the upper back. Several microbe-metabolite associations were uncovered, including Cutibacterium, detected in the upper back area, which was correlated with the distribution of MUFAs, and Anaerococcus spp. found in the underarms, which were associated with a series of different bile acids. Overall, these findings point to a notable impact of the silver-threaded material on the skin microbiome and chemistry. We observed that relatively subtle changes in the microbiome result in pronounced shifts in molecular composition. IMPORTANCE The impact of silver-threaded material on human skin chemistry and microbiome is largely unknown. Although the most abundant taxa remained unaffected, silver caused an increase in diversity and richness of low-abundant bacteria and a decrease in chemical diversity. The major change was an increase in the abundance of various monounsaturated fatty acids that were also correlated with Cutibacterium. Additionally, Anaerococcus spp., found in the underarms, were associated with different bile acids in the armpit samples. Overall, the impact of the silver-threaded clothing was gender and body site specific.

The molecular impact of life in an indoor environment.

The chemistry of indoor surfaces and the role of microbes in shaping and responding to that chemistry are largely unexplored. We found that, over 1 month, people's presence and activities profoundly reshaped the chemistry of a house. Molecules associated with eating/cooking, bathroom use, and personal care were found throughout the entire house, while molecules associated with medications, outdoor biocides, and microbially derived compounds were distributed in a location-dependent manner. The house and its microbial occupants, in turn, also introduced chemical transformations such as oxidation and transformations of foodborne molecules. The awareness of and the ability to observe the molecular changes introduced by people should influence future building designs.

Microbial and Nonvolatile Chemical Diversities of Chinese Dark Teas Are Differed by Latitude and Pile Fermentation.

Understanding the microbial and chemical diversities, as well as what affects these diversities, is important for modern manufacturing of traditional fermented foods. In this work, Chinese dark teas (CDTs) that are traditional microbial fermented beverages with relatively high sample diversity were collected. Microbial DNA amplicon sequencing and mass spectrometry-based untargeted metabolomics show that the CDT microbial ß diversity, as well as the nonvolatile chemical α and ß diversities, is determined by the primary impact factors of geography and manufacturing procedures, in particular, latitude and pile fermentation after blending. A large number of metabolites sharing between CDTs and fungi were discovered by Feature-based Molecular Networking (FBMN) on the Global Natural Products Social Molecular Networking (GNPS) web platform. These molecules, such as prenylated cyclic dipeptides and B-vitamins, are functionally important for nutrition, biofunctions, and flavor. Molecular networking has revealed patterns in metabolite profiles on a chemical family level in addition to individual structures.

Metabolic Profiling of Interspecies Interactions During Sessile Bacterial Cultivation Reveals Growth and Sporulation Induction in Paenibacillus amylolyticus in Response to Xanthomonas retroflexus.

The toolbox available for microbiologists to study interspecies interactions is rapidly growing, and with continuously more advanced instruments, we are able to expand our knowledge on establishment and function of microbial communities. However, unravelling molecular interspecies interactions in complex biological systems remains a challenge, and interactions are therefore often studied in simplified communities. Here we perform an in-depth characterization of an observed interspecies interaction between two co-isolated bacteria, Xanthomonas retroflexus and Paenibacillus amylolyticus. Using microsensor measurements for mapping the chemical environment, we show how X. retroflexus promoted an alkalization of its local environment through degradation of amino acids and release of ammonia. When the two species were grown in proximity, the modified local environment induced a morphological change and growth of P. amylolyticus followed by sporulation. 2D spatial metabolomics enabled visualization and mapping of the degradation of oligopeptide structures by X. retroflexus and morphological changes of P. amylolyticus through e.g. the release of membrane-associated metabolites. Proteome analysis and microscopy were used to validate the shift from vegetative growth towards sporulation. In summary, we demonstrate how environmental profiling by combined application of microsensor, microscopy, metabolomics and proteomics approaches can reveal growth and sporulation promoting effects resulting from interspecies interactions.

Functional genomics and metabolomics advance the ethnobotany of the Samoan traditional medicine "matalafi".

The leaf homogenate of Psychotria insularum is widely used in Samoan traditional medicine to treat inflammation associated with fever, body aches, swellings, wounds, elephantiasis, incontinence, skin infections, vomiting, respiratory infections, and abdominal distress. However, the bioactive components and underlying mechanisms of action are unknown. We used chemical genomic analyses in the model organism Saccharomyces cerevisiae (baker's yeast) to identify and characterize an iron homeostasis mechanism of action in the traditional medicine as an unfractionated entity to emulate its traditional use. Bioactivity-guided fractionation of the homogenate identified two flavonol glycosides, rutin and nicotiflorin, each binding iron in an ion-dependent molecular networking metabolomics analysis. Translating results to mammalian immune cells and traditional application, the iron chelator activity of the P. insularum homogenate or rutin decreased proinflammatory and enhanced anti-inflammatory cytokine responses in immune cells. Together, the synergistic power of combining traditional knowledge with chemical genomics, metabolomics, and bioassay-guided fractionation provided molecular insight into a relatively understudied Samoan traditional medicine and developed methodology to advance ethnobotany.

B. infantis EVC001 Is Well-Tolerated and Improves Human Milk Oligosaccharide Utilization in Preterm Infants in the Neonatal Intensive Care Unit.

Not all infants carry specialized gut microbes, meaning they cannot digest human milk oligosaccharides and therefore do not receive complete benefits from human milk. B. infantis EVC001 is equipped to convert the full array of complex oligosaccharides into compounds usable by the infant, making it an ideal candidate to stabilize gut function and improve nutrition in preterm infants. A prospective, open-label study design was used to evaluate the tolerability of B. infantis EVC001 and its effects on the fecal microbiota in preterm infants in a Neonatal Intensive Care Unit. Thirty preterm infants <1,500 g and/or <33 weeks gestation at birth were divided into two matched groups, and control infants were enrolled and discharged prior to enrolling EVC001 infants to prevent cross-colonization of B. infantis: (1) fifteen control infants received no EVC001, and (2) fifteen infants received once-daily feedings of B. infantis EVC001 (8.0 x 109 CFU) in MCT oil. Clinical information regarding medications, growth, nutrition, gastrointestinal events, diagnoses, and procedures was collected throughout admission. Infant stool samples were collected at baseline, Study Days 14 and 28, and 34-, 36-, and 38-weeks of gestation. Taxonomic composition of the fecal microbiota, functional microbiota analysis, B. infantis, and human milk oligosaccharides (HMOs) in the stool were determined or quantified using 16S rRNA gene sequencing, metagenomic sequencing, qPCR, and mass spectrometry, respectively. No adverse events or tolerability issues related to EVC001 were reported. Control infants had no detectable levels of B. infantis. EVC001 infants achieved high levels of B. infantis (mean = 9.7 Log10 CFU/µg fecal DNA) by Study Day 14, correlating with less fecal HMOs (ρ = -0.83, P < 0.0001), indicating better HMO utilization in the gut. In this study, B. infantis EVC001 was shown to be safe, well-tolerated, and efficient in colonizing the preterm infant gut and able to increase the abundance of bifidobacteria capable of metabolizing HMOs, resulting in significantly improved utilization of human milk. Clinical Trial Registration: https://clinicaltrials.gov/ct2/show/NCT03939546, identifier: NCT03939546.

Auto-deconvolution and molecular networking of gas chromatography-mass spectrometry data.

We engineered a machine learning approach, MSHub, to enable auto-deconvolution of gas chromatography-mass spectrometry (GC-MS) data. We then designed workflows to enable the community to store, process, share, annotate, compare and perform molecular networking of GC-MS data within the Global Natural Product Social (GNPS) Molecular Networking analysis platform. MSHub/GNPS performs auto-deconvolution of compound fragmentation patterns via unsupervised non-negative matrix factorization and quantifies the reproducibility of fragmentation patterns across samples.

A Cutibacterium acnes antibiotic modulates human skin microbiota composition in hair follicles.

The composition of the skin microbiota varies widely among individuals when sampled at the same body site. A key question is which molecular factors determine strain-level variability within sub-ecosystems of the skin microbiota. Here, we used a genomics-guided approach to identify an antibacterial biosynthetic gene cluster in Cutibacterium acnes (formerly Propionibacterium acnes), a human skin commensal bacterium that is widely distributed across individuals and skin sites. Experimental characterization of this biosynthetic gene cluster resulted in identification of a new thiopeptide antibiotic, cutimycin. Analysis of individual human skin hair follicles revealed that cutimycin contributed to the ecology of the skin hair follicle microbiota and helped to reduce colonization of skin hair follicles by Staphylococcus species.

ReDU: a framework to find and reanalyze public mass spectrometry data.

We present ReDU ( https://redu.ucsd.edu/ ), a system for metadata capture of public mass spectrometry-based metabolomics data, with validated controlled vocabularies. Systematic capture of knowledge enables the reanalysis of public data and/or co-analysis of one's own data. ReDU enables multiple types of analyses, including finding chemicals and associated metadata, comparing the shared and different chemicals between groups of samples, and metadata-filtered, repository-scale molecular networking.

Feature-Based Molecular Networking Analysis of the Metabolites Produced by In Vitro Solid-State Fermentation Reveals Pathways for the Bioconversion of Epigallocatechin Gallate.

Dark teas are prepared by a microbial fermentation process. Flavan-3-ol B-ring fission analogues (FBRFAs) are some of the key bioactive constituents that characterize dark teas. The precursors and the synthetic mechanism involved in the formation of FBRFAs are not known. Using a unique solid-state fermentation system with ß-cyclodextrin inclusion complexation as well as targeted chromatographic isolation, spectroscopic identification, and Feature-based Molecular Networking on the Global Natural Products Social Molecular Networking web platform, we reveal that dihydromyricetin and the FBRFAs, including teadenol A and fuzhuanin A, are derived from epigallocatechin gallate upon exposure to fungal strains isolated from Fuzhuan brick tea. In particular, the strains from subphylum Pezizomycotina were key drivers for these B-/C-ring oxidation transformations. These are the same transformations seen during the fermentation process of dark teas. These discoveries set the stage to enrich dark teas and other food products for these health-promoting constituents.

Global chemical effects of the microbiome include new bile-acid conjugations.

A mosaic of cross-phylum chemical interactions occurs between all metazoans and their microbiomes. A number of molecular families that are known to be produced by the microbiome have a marked effect on the balance between health and disease1-9. Considering the diversity of the human microbiome (which numbers over 40,000 operational taxonomic units10), the effect of the microbiome on the chemistry of an entire animal remains underexplored. Here we use mass spectrometry informatics and data visualization approaches11-13 to provide an assessment of the effects of the microbiome on the chemistry of an entire mammal by comparing metabolomics data from germ-free and specific-pathogen-free mice. We found that the microbiota affects the chemistry of all organs. This included the amino acid conjugations of host bile acids that were used to produce phenylalanocholic acid, tyrosocholic acid and leucocholic acid, which have not previously been characterized despite extensive research on bile-acid chemistry14. These bile-acid conjugates were also found in humans, and were enriched in patients with inflammatory bowel disease or cystic fibrosis. These compounds agonized the farnesoid X receptor in vitro, and mice gavaged with the compounds showed reduced expression of bile-acid synthesis genes in vivo. Further studies are required to confirm whether these compounds have a physiological role in the host, and whether they contribute to gut diseases that are associated with microbiome dysbiosis.

Untargeted mass spectrometry-based metabolomics approach unveils molecular changes in raw and processed foods and beverages.

In our daily lives, we consume foods that have been transported, stored, prepared, cooked, or otherwise processed by ourselves or others. Food storage and preparation have drastic effects on the chemical composition of foods. Untargeted mass spectrometry analysis of food samples has the potential to increase our chemical understanding of these processes by detecting a broad spectrum of chemicals. We performed a time-based analysis of the chemical changes in foods during common preparations, such as fermentation, brewing, and ripening, using untargeted mass spectrometry and molecular networking. The data analysis workflow presented implements an approach to study changes in food chemistry that can reveal global alterations in chemical profiles, identify changes in abundance, as well as identify specific chemicals and their transformation products. The data generated in this study are publicly available, enabling the replication and re-analysis of these data in isolation, and serve as a baseline dataset for future investigations.

Molecular and Microbial Microenvironments in Chronically Diseased Lungs Associated with Cystic Fibrosis.

To visualize the personalized distributions of pathogens and chemical environments, including microbial metabolites, pharmaceuticals, and their metabolic products, within and between human lungs afflicted with cystic fibrosis (CF), we generated three-dimensional (3D) microbiome and metabolome maps of six explanted lungs from three cystic fibrosis patients. These 3D spatial maps revealed that the chemical environments differ between patients and within the lungs of each patient. Although the microbial ecosystems of the patients were defined by the dominant pathogen, their chemical diversity was not. Additionally, the chemical diversity between locales in the lungs of the same individual sometimes exceeded interindividual variation. Thus, the chemistry and microbiome of the explanted lungs appear to be not only personalized but also regiospecific. Previously undescribed analogs of microbial quinolones and antibiotic metabolites were also detected. Furthermore, mapping the chemical and microbial distributions allowed visualization of microbial community interactions, such as increased production of quorum sensing quinolones in locations where Pseudomonas was in contact with Staphylococcus and Granulicatella, consistent with in vitro observations of bacteria isolated from these patients. Visualization of microbe-metabolite associations within a host organ in early-stage CF disease in animal models will help elucidate the complex interplay between the presence of a given microbial structure, antibiotics, metabolism of antibiotics, microbial virulence factors, and host responses.IMPORTANCE Microbial infections are now recognized to be polymicrobial and personalized in nature. Comprehensive analysis and understanding of the factors underlying the polymicrobial and personalized nature of infections remain limited, especially in the context of the host. By visualizing microbiomes and metabolomes of diseased human lungs, we reveal how different the chemical environments are between hosts that are dominated by the same pathogen and how community interactions shape the chemical environment or vice versa. We highlight that three-dimensional organ mapping methods represent hypothesis-building tools that allow us to design mechanistic studies aimed at addressing microbial responses to other microbes, the host, and pharmaceutical drugs.

The impact of skin care products on skin chemistry and microbiome dynamics.

BACKGROUND: Use of skin personal care products on a regular basis is nearly ubiquitous, but their effects on molecular and microbial diversity of the skin are unknown. We evaluated the impact of four beauty products (a facial lotion, a moisturizer, a foot powder, and a deodorant) on 11 volunteers over 9 weeks. RESULTS: Mass spectrometry and 16S rRNA inventories of the skin revealed decreases in chemical as well as in bacterial and archaeal diversity on halting deodorant use. Specific compounds from beauty products used before the study remain detectable with half-lives of 0.5-1.9 weeks. The deodorant and foot powder increased molecular, bacterial, and archaeal diversity, while arm and face lotions had little effect on bacterial and archaeal but increased chemical diversity. Personal care product effects last for weeks and produce highly individualized responses, including alterations in steroid and pheromone levels and in bacterial and archaeal ecosystem structure and dynamics. CONCLUSIONS: These findings may lead to next-generation precision beauty products and therapies for skin disorders.