Robert A. Quinn

Microbiome-wide association studies link dynamic microbial consortia to disease

Rapid advances in DNA sequencing, metabolomics, proteomics and computational tools are dramatically increasing access to the microbiome and identification of its links with disease. In particular, time-series studies and multiple molecular perspectives are facilitating microbiome-wide association studies, which are analogous to genome-wide association studies. Early findings point to actionable outcomes of microbiome-wide association studies, although their clinical application has yet to be approved. An appreciation of the complexity of interactions among the microbiome and the hosts diet, chemistry and health, as well as determining the frequency of observations that are needed to capture and integrate this dynamic interface, is paramount for developing precision diagnostics and therapies that are based on the microbiome.

Illuminating the dark matter in metabolomics

Despite the over 100-y history of mass spectrometry, it remains challenging to link the large volume of known chemical structures to the data obtained with mass spectrometers. Presently, only 1.8% of spectra in an untargeted metabolomics experiment can be annotated. This means that the vast majority of information collected by metabolomics is “dark matter,” chemical signatures that remain uncharacterized (Fig. 1). For a genomic comparison, 80% of predicted genes in the Escherichia coli genome are known. In a bacteriophage metagenome, a well-known frontier of biological dark matter, the amount of known genes is 1–30%, depending on the sample (1). Thus, one could argue that we know more about the genetics of uncultured phage than we do about the chemistry within our own bodies. Much of the chemical dark matter may include known structures, but they remain undiscovered because the reference spectra are not available in mass spectrometry databases. The only way to overcome this challenge is through the development of computational solutions. In PNAS, Duhrkop et al. describe the development of such a computational tool, called CSI (compound structure identification):FingerID (2). The tool is designed to aid in the annotation of chemistries that can be observed by mass spectrometry. CSI:FingerID uses fragmentation trees to connect tandem MS (MS/MS) data to chemical structures found in public chemistry databases. Tools such as this can allow metabolomics with mass spectrometry to become as commonly used and scientifically productive as sequencing technologies have in the field of genomics.

Real-time metabolomics on living microorganisms using ambient electrospray ionization flow-probe.

Microorganisms such as bacteria and fungi produce a variety of specialized metabolites that are invaluable for agriculture, biological research, and drug discovery. However, the screening of microbial metabolic output is usually a time-intensive task. Here, we utilize a liquid microjunction surface sampling probe for electrospray ionization-mass spectrometry to extract and ionize metabolite mixtures directly from living microbial colonies grown on soft nutrient agar in Petri-dishes without any sample pretreatment. To demonstrate the robustness of the method, this technique was applied to observe the metabolic output of more than 30 microorganisms, including yeast, filamentous fungi, pathogens, and marine-derived bacteria, that were collected worldwide. Diverse natural products produced from different microbes, including Streptomyces coelicolor , Bacillus subtilis , and Pseudomonas aeruginosa are further characterized.

Breath gas metabolites and bacterial metagenomes from cystic fibrosis airways indicate active pH neutral 2,3-butanedione fermentation

The airways of cystic fibrosis (CF) patients are chronically colonized by patient-specific polymicrobial communities. The conditions and nutrients available in CF lungs affect the physiology and composition of the colonizing microbes. Recent work in bioreactors has shown that the fermentation product 2,3-butanediol mediates cross-feeding between some fermenting bacteria and Pseudomonas aeruginosa, and that this mechanism increases bacterial current production. To examine bacterial fermentation in the respiratory tract, breath gas metabolites were measured and several metagenomes were sequenced from CF and non-CF volunteers. 2,3-butanedione was produced in nearly all respiratory tracts. Elevated levels in one patient decreased during antibiotic treatment, and breath concentrations varied between CF patients at the same time point. Some patients had high enough levels of 2,3-butanedione to irreversibly damage lung tissue. Antibiotic therapy likely dictates the activities of 2,3-butanedione-producing microbes, which suggests a need for further study with larger sample size. Sputum microbiomes were dominated by P. aeruginosa, Streptococcus spp. and Rothia mucilaginosa, and revealed the potential for 2,3-butanedione biosynthesis. Genes encoding 2,3-butanedione biosynthesis were disproportionately abundant in Streptococcus spp, whereas genes for consumption of butanedione pathway products were encoded by P. aeruginosa and R. mucilaginosa. We propose a model where low oxygen conditions in CF lung lead to fermentation and a decrease in pH, triggering 2,3-butanedione fermentation to avoid lethal acidification. We hypothesize that this may also increase phenazine production by P. aeruginosa, increasing reactive oxygen species and providing additional electron acceptors to CF microbes.

Biogeochemical Forces Shape the Composition and Physiology of Polymicrobial Communities in the Cystic Fibrosis Lung

ABSTRACT The cystic fibrosis (CF) lung contains thick mucus colonized by opportunistic pathogens which adapt to the CF lung environment over decades. The difficulty associated with sampling airways has impeded a thorough examination of the biochemical microhabitats these pathogens are exposed to. An indirect approach is to study the responses of microbial communities to these microhabitats, facilitated by high-throughput sequencing of microbial DNA and RNA from sputum samples. Microbial metagenomes and metatranscriptomes were sequenced from multiple CF patients, and the reads were assigned taxonomy and function through sequence homology to NCBI and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database hierarchies. For a comparison, saliva microbial metagenomes from the Human Microbiome Project (HMP) were also analyzed. These analyses identified that functions encoded and expressed by CF microbes were significantly enriched for amino acid catabolism, folate biosynthesis, and lipoic acid biosynthesis. The data indicate that the community uses oxidative phosphorylation as a major energy source but that terminal electron acceptors were diverse. Nitrate reduction was the most abundant anaerobic respiratory pathway, and genes for nitrate reductase were largely assigned to Pseudomonas and Rothia. Although many reductive pathways of the nitrogen cycle were present, the cycle was incomplete, because the oxidative pathways were absent. Due to the abundant amino acid catabolism and incomplete nitrogen cycle, the CF microbial community appears to accumulate ammonia. This finding was verified experimentally using a CF bronchiole culture model system. The data also revealed abundant sensing and transport of iron, ammonium, zinc, and other metals along with a low-oxygen environment. This study reveals the core biochemistry and physiology of the CF microbiome. IMPORTANCE The cystic fibrosis (CF) microbial community is complex and adapts to the environmental conditions of the lung over the lifetime of a CF patient. This analysis illustrates the core functions of the CF microbial community in the context of CF lung biochemistry. There are many studies of the metabolism and physiology of individual microbes within the CF lung, but none that collectively analyze data from the whole microbiome. Understanding the core metabolism of microbes that inhabit the CF lung can provide new targets for novel therapies. The fundamental processes that CF pathogens rely on for survival may represent an Achilles heel for this pathogenic community. Novel therapies that are designed to disrupt understudied survival strategies of the CF microbial community may succeed against otherwise untreatable or antibiotic-resistant microbes. The cystic fibrosis (CF) microbial community is complex and adapts to the environmental conditions of the lung over the lifetime of a CF patient. This analysis illustrates the core functions of the CF microbial community in the context of CF lung biochemistry. There are many studies of the metabolism and physiology of individual microbes within the CF lung, but none that collectively analyze data from the whole microbiome. Understanding the core metabolism of microbes that inhabit the CF lung can provide new targets for novel therapies. The fundamental processes that CF pathogens rely on for survival may represent an Achilles heel for this pathogenic community. Novel therapies that are designed to disrupt understudied survival strategies of the CF microbial community may succeed against otherwise untreatable or antibiotic-resistant microbes.

The Upper Respiratory Tract as a Microbial Source for Pulmonary Infections in Cystic Fibrosis. Parallels from Island Biogeography

A continuously mixed series of microbial communities inhabits various points of the respiratory tract, with community composition determined by distance from colonization sources, colonization rates, and extinction rates. Ecology and evolution theory developed in the context of biogeography is relevant to clinical microbiology and could reframe the interpretation of recent studies comparing communities from lung explant samples, sputum samples, and oropharyngeal swabs. We propose an island biogeography model of the microbial communities inhabiting different niches in human airways. Island biogeography as applied to communities separated by time and space is a useful parallel for exploring microbial colonization of healthy and diseased lungs, with the potential to inform our understanding of microbial community dynamics and the relevance of microbes detected in different sample types. In this perspective, we focus on the intermixed microbial communities inhabiting different regions of the airways of patients with cystic fibrosis.

Bacterial Communities Associated with Lesions of Shell Disease in the American Lobster, Homarus americanus Milne-Edwards

ABSTRACT Shell disease in Crustacea is a widely recognized syndrome having a polymicrobial etiology, and manifesting itself as lesions of the shell with a variable structure and shell location. We characterized major members of bacterial communities in epizootic shell disease lesions of the American lobster (Homarus americanus, Milne Edwards) and compared these communities with the ones found in study cases of impoundment and enzootic shell disease. Bacteria belonging to several Flavobacteriaceae genera (Aquimarina, Tenacibaculum, Polaribacter, Maribacter, Cellulophaga) within the phylum Bacteroidetes appear to have particular attraction to lobster lesions. The most prominent Bacteroidetes in lobster lesions were representatives of the genus Aquimarina sp., but only Aquimarina ‘homaria’ was detected in all analyzed lesions of epizootic, impoundment, and enzootic shell disease. It was found on 45% of surfaces unaffected by shell disease, but in smaller numbers compared with lesions. Alphaproteobacteria represent the most diverse class of proteobacteria found in both lesions and on unaffected surfaces. Three bacteria of this class appear to be ubiquitous in shell disease lesions, but only one specific alphaproteobacterium tentatively assigned to the genus Thalassobius (herein designated as ‘Thalassobius’ sp.) was present in all analyzed lesions of epizootic, impoundment, and enzootic shell disease. A ubiquitous gammaproteobacterium called ‘Candidatus Homarophilus dermatus’ was also prevalent in lesions, but just as commonly it was associated with surfaces unaffected by shell disease. The bacteria A. ‘homaria’ and ‘Thalassobius’ sp. are dominant and appear obligatory in lobster shell lesions, and are only occasionally detected on unaffected surfaces, which serve as intermediate reservoirs for the two potential pathogens. Therefore, these two bacteria stand out as potential shell-disease pathogens.

Exposures of Homarus americanus Shell to Three Bacteria Isolated from Naturally Occurring Epizootic Shell Disease Lesions

ABSTRACT Epizootic shell disease (ESD) is an emerging form of shell disease of the American lobster (Homarus americanus) that has had detrimental effects on the fishery in southern New England. Three bacteria commonly isolated from lesions of wild lobsters with ESD—a novel Aquimarina sp. (A. ‘homaria’ I32.4), a novel Rhodobacteraceae species (‘Thalassobius’ sp. I31.1) and a Pseudoalteromonas sp. (Pseudoalteromonas ‘gracilis’ ISA7.3)—were applied directly to normal and abraded juvenile lobster carapaces, and then monitored for persistence over time and for the development of shell-disease lesions at 3 different temperatures (10°C, 15°C, and 20°C). Without abrasion of the carapace, no lesions developed in the exposures. After abrasion and exposure with a pure culture of A. ‘homaria’ I32.4, lesions developed at all 3 temperature and A. ‘homaria’ was detected in the lesions of all animals tested. Surprisingly, ‘Thalassobius’ sp. I31.1 also colonized these lesions. A coexposure with all 3 bacteria also demonstrated lesion development and the persistence of A. ‘homaria’ I32.4 and ‘Thalassobius’ sp. I31.1. The bacterium P. ‘gracilis’ ISA7.3 was not able to persist in any of the challenged lesions. Abraded areas of the cuticle with no bacteria added directly were also colonized by A. ‘homaria’ and ‘Thalassobius’ sp., and moderate lesions developed; however, the directly exposed lesions were significantly more severe (P < 0.05). The bacterium A. ‘homaria’, but not ‘Thalassobius’ sp., was detected in spontaneous lesions that developed independent of any abrasion and/or bacterial exposures. A novel bacterium, ‘Candidatus Kopriimonas aquarianus’ was also detected in spontaneous lesions. This study shows that 2 bacteria isolated from ESD lesions of wild lobsters are able to persist in and act together as important components of lesion development on abraded surfaces of American lobsters. This indicates that they are likely major contributors to lesion development in the ESD polymicrobial infection and may represent significant pathogens of the American lobster.

Microbial, host and xenobiotic diversity in the cystic fibrosis sputum metabolome.

Cystic fibrosis (CF) lungs are filled with thick mucus that obstructs airways and facilitates chronic infections. Pseudomonas aeruginosa is a significant pathogen of this disease that produces a variety of toxic small molecules. We used molecular networking-based metabolomics to investigate the chemistry of CF sputa and assess how the microbial molecules detected reflect the microbiome and clinical culture history of the patients. Metabolites detected included xenobiotics, P. aeruginosa specialized metabolites and host sphingolipids. The clinical culture and microbiome profiles did not correspond to the detection of P. aeruginosa metabolites in the same samples. The P. aeruginosa molecules that were detected in sputum did not match those from laboratory cultures. The pseudomonas quinolone signal (PQS) was readily detectable from cultured strains, but absent from sputum, even when its precursor molecules were present. The lack of PQS production in vivo is potentially due to the chemical nature of the CF lung environment, indicating that culture-based studies of this pathogen may not explain its behavior in the lung. The most differentially abundant molecules between CF and non-CF sputum were sphingolipids, including sphingomyelins, ceramides and lactosylceramide. As these highly abundant molecules contain the inflammatory mediator ceramide, they may have a significant role in CF hyperinflammation. This study demonstrates that the chemical makeup of CF sputum is a complex milieu of microbial, host and xenobiotic molecules. Detection of a bacterium by clinical culturing and 16S rRNA gene profiling do not necessarily reflect the active production of metabolites from that bacterium in a sputum sample.

Molecular Networking As a Drug Discovery, Drug Metabolism, and Precision Medicine Strategy

Molecular networking is a tandem mass spectrometry (MS/MS) data organizational approach that has been recently introduced in the drug discovery, metabolomics, and medical fields. The chemistry of molecules dictates how they will be fragmented by MS/MS in the gas phase and, therefore, two related molecules are likely to display similar fragment ion spectra. Molecular networking organizes the MS/MS data as a relational spectral network thereby mapping the chemistry that was detected in an MS/MS-based metabolomics experiment. Although the wider utility of molecular networking is just beginning to be recognized, in this review we highlight the principles behind molecular networking and its use for the discovery of therapeutic leads, monitoring drug metabolism, clinical diagnostics, and emerging applications in precision medicine.