Muramidases found in the foregut microbiome of the Tammar wallaby can direct cell aggregation and biofilm formation

We describe here the role of muramidases present in clones of metagenomic DNA that result in cell aggregation and biofilm formation by Escherichia coli. The metagenomic clones were obtained from uncultured Lachnospiraceae-affiliated bacteria resident in the foregut microbiome of the Tammar wallaby. One of these fosmid clones (p49C2) was chosen for more detailed studies and a variety of genetic methods were used to delimit the region responsible for the phenotype to an open reading frame of 1425 bp. Comparative sequence analysis with other fosmid clones giving rise to the same phenotype revealed the presence of muramidase homologues with the same modular composition. Phylogenetic analysis of the fosmid sequence data assigned these fosmid inserts to recently identified, but uncultured, phylogroups of Lachnospiraceae believed to be numerically dominant in the foregut microbiome of the Tammar wallaby. The muramidase is a modular protein containing putative N-acetylmuramoyl--alanine amidase and an endo-β-N-acetylglucosaminidase catalytic module, with a similar organization and functional properties to some Staphylococcal autolysins that also confer adhesive properties and biofilm formation. We also show here that the cloned muramidases result in the production of extracellular DNA, which appears to be the key for biofilm formation and autoaggregation. Collectively, these findings suggest that biofilm formation and cell aggregation in gut microbiomes might occur via the concerted action of carbohydrate-active enzymes and the production of extracellular DNA to serve as a biofilm scaffold.

The intestinal microbiome in human disease and how it relates to arthritis and spondyloarthritis

Humans and microbes have developed a symbiotic relationship over time, and alterations in this symbiotic relationship have been linked to several immune mediated diseases such as inflammatory bowel disease, type 1 diabetes and spondyloarthropathies. Improvements in sequencing technologies, coupled with a renaissance in 16S rRNA gene based community profiling, have enabled the characterization of microbiomes throughout the body including the gut. Improved characterization and understanding of the human gut microbiome means the gut flora is progressively being explored as a target for novel therapies including probiotics and faecal microbiota transplants. These innovative therapies are increasingly used for patients with debilitating conditions where conventional treatments have failed. This review discusses the current understanding of the interplay between host genetics and the gut microbiome in the pathogenesis of spondyloarthropathies, and how this may relate to potential therapies for these conditions.

Does the microbiome play a causal role in spondyloarthritis?

The purpose of this study is to review the potential causal role of the microbiome in the pathogenesis of spondyloarthritis. The method used for the study is literature review. The microbiome plays a major role in educating the immune response. The microbiome is strongly implicated in inflammatory bowel disease which has clinical and genetic overlap with spondyloarthritis. The microbiome also plays a causal role in bowel and joint disease in HLA B27/human beta 2 microglobulin transgenic rats. The mechanism(s) by which HLA B27 could influence the microbiome is unknown but theories include an immune response gene selectivity, an effect on dendritic cell function, or a mucosal immunodeficiency. Bacteria are strongly implicated in the pathogenesis of spondyloarthritis. Studies to understand how HLA B27 affects bacterial ecosystems should be encouraged.

Breastmilk-saliva interactions boost innate immunity by regulating the oral microbiome in early infancy

Introduction Xanthine oxidase (XO) is distributed in mammals largely in the liver and small intestine, but also is highly active in milk where it generates hydrogen peroxide (H2O2). Adult human saliva is low in hypoxanthine and xanthine, the substrates of XO, and high in the lactoperoxidase substrate thiocyanate, but saliva of neonates has not been examined. Results Median concentrations of hypoxanthine and xanthine in neonatal saliva (27 and 19 μM respectively) were ten-fold higher than in adult saliva (2.1 and 1.7 μM). Fresh breastmilk contained 27.3±12.2 μM H2O2 but mixing baby saliva with breastmilk additionally generated >40 μM H2O2, sufficient to inhibit growth of the opportunistic pathogens Staphylococcus aureus and Salmonella spp. Oral peroxidase activity in neonatal saliva was variable but low (median 7 U/L, range 2–449) compared to adults (620 U/L, 48–1348), while peroxidase substrate thiocyanate in neonatal saliva was surprisingly high. Baby but not adult saliva also contained nucleosides and nucleobases that encouraged growth of the commensal bacteria Lactobacillus, but inhibited opportunistic pathogens; these nucleosides/bases may also promote growth of immature gut cells. Transition from neonatal to adult saliva pattern occurred during the weaning period. A survey of saliva from domesticated mammals revealed wide variation in nucleoside/base patterns. Discussion and Conclusion During breast-feeding, baby saliva reacts with breastmilk to produce reactive oxygen species, while simultaneously providing growth-promoting nucleotide precursors. Milk thus plays more than a simply nutritional role in mammals, interacting with infant saliva to produce a potent combination of stimulatory and inhibitory metabolites that regulate early oral–and hence gut–microbiota. Consequently, milk-saliva mixing appears to represent unique biochemical synergism which boosts early innate immunity.

Rumen microbial community composition varies with diet and host, but a core microbiome is found across a wide geographical range

Ruminant livestock are important sources of human food and global greenhouse gas emissions. Feed degradation and methane formation by ruminants rely on metabolic interactions between rumen microbes and affect ruminant productivity. Rumen and camelid foregut microbial community composition was determined in 742 samples from 32 animal species and 35 countries, to estimate if this was influenced by diet, host species, or geography. Similar bacteria and archaea dominated in nearly all samples, while protozoal communities were more variable. The dominant bacteria are poorly characterised, but the methanogenic archaea are better known and highly conserved across the world. This universality and limited diversity could make it possible to mitigate methane emissions by developing strategies that target the few dominant methanogens. Differences in microbial community compositions were predominantly attributable to diet, with the host being less influential. There were few strong co-occurrence patterns between microbes, suggesting that major metabolic interactions are non-selective rather than specific. © 2015 Macmillan Publishers Limited.

Production of Carcinogenic Acetaldehyde by Oral Microbiome

Oral cancer is the seventh most common cancer worldwide and its incidence is increasing. The most important risk factors for oral cancer are chronic alcohol consumption and tobacco smoking, up to 80 % of oral carcinomas are estimated to be caused by alcohol and tobacco. They both trigger an increased level of salivary acetaldehyde, during and after consumption, which is believed to lead to carcinogenesis. Acetaldehyde has multiple mutagenic features and it has recently been classified as a Group 1 carcinogen for humans by the International Agency for Research on Cancer. Acetaldehyde is metabolized from ethanol by microbes of oral microbiota. Some oral microbes possess alcohol dehydrogenase enzyme (ADH) activity, which is the main enzyme in acetaldehyde production. Many microbes are also capable of acetaldehyde production via alcohol fermentation from glucose. However, metabolism of ethanol into acetaldehyde leads to production of high levels of this carcinogen. Acetaldehyde is found in saliva during and after alcohol consumption. In fact, rather low ethanol concentrations (2-20mM) derived from blood to saliva are enough for microbial acetaldehyde production. The high acetaldehyde levels in saliva after alcohol challenge are explained by the lack of oral microbiota and mucosa to detoxify acetaldehyde by metabolizing it into acetate and acetyl coenzymeA. The aim of this thesis project was to specify the role of oral microbes in the in vitro production of acetaldehyde in the presence of ethanol. In addition, it was sought to establish whether microbial metabolism could also produce acetaldehyde from glucose. Furthermore, the potential of xylitol to inhibit ethanol metabolism and acetaldehyde production was explored. Isolates of oral microbes were used in the first three studies. Acetaldehyde production was analyzed after ethanol, glucose and fructose incubation with gas chromatography measurement. In studies I and III, the ADH enzyme activity of some microbes was measured by fluorescence. The effect of xylitol was analyzed by incubating microbes with ethanol and xylitol. The fourth study was made ex vivo and microbial samples obtained from different patient groups were analyzed. This work has demonstrated that isolates of oral microbiota are able to produce acetaldehyde in the presence of clinically relevant ethanol and glucose concentrations. Significant differences were found between microbial species and isolates from different patient groups. In particular, the ability of candidal isolates from APECED patients to produce significantly more acetaldehyde in glucose incubation compared to healthy and cancer patient isolates is an interesting observation. Moreover, xylitol was found to reduce their acetaldehyde production significantly. Significant ADH enzyme activity was found in the analyzed high acetaldehyde producing streptococci and candida isolates. In addition, xylitol was found to reduce the ADH enzyme activity of C. albicans. Some results from the ex vivo study were controversial, since acetaldehyde production did not correlate as expected with the amount of microbes in the samples. Nevertheless, the samples isolated from patients did produce significant amounts of acetaldehyde with a clinically relevant ethanol concentration.

Functional metagenomic analysis of the human gut microbiome to identify novel salt tolerance genes

The ability to adapt to and respond to increases in external osmolarity is an important characteristic that enables bacteria to survive and proliferate in different environmental niches. When challenged with increased osmolarity, due to sodium chloride (NaCl) for example, bacteria elicit a phased response; firstly via uptake of potassium (K+), which is known as the primary response. This primary response is followed by the secondary response which is characterised by the synthesis or uptake of compatible solutes (osmoprotectants). The overall osmotic stress response is much broader however, involving many diverse cellular systems and processes. These ancillary mechanisms are arguably more interesting and give a more complete view of the osmotic stress response. The aim of this thesis was to identify novel genetic loci from the human gut microbiota that confer increased tolerance to osmotic stress using a functional metagenomic approach. Functional metagenomics is a powerful tool that enables the identification of novel genes from as yet uncultured bacteria from diverse environments through cloning, heterologous expression and phenotypic identification of a desired trait. Functional metagenomics does not rely on any previous sequence information to known genes and can therefore enable the discovery of completely novel genes and assign functions to new or known genes. Using a functional metagenomic approach, we have assigned a novel function to previously annotated genes; murB, mazG and galE, as well as a putative brp/blh family beta-carotene 15,15’-monooxygenase. Finally, we report the identification of a completely novel salt tolerance determinant with no current known homologues in the databases. Overall the genes identified originate from diverse taxonomic and phylogenetic groups commonly found in the human gastrointestinal (GI) tract, such as Collinsella and Eggerthella, Akkermansia and Bacteroides from the phyla Actinobacteria, Verrucomicrobia and Bacteroidetes, respectively. In addition, a number of the genes appear to have been acquired via lateral gene transfer and/or encoded on a prophage. To our knowledge, this thesis represents the first investigation to identify novel genes from the human gut microbiota involved in the bacterial osmotic stress response.

The airway microbiome in cystic fibrosis: challenges for therapy

Although cystic fibrosis pulmonary infection is polymicrobial, routine laboratory methods focus on the detection of a small number of known pathogens. Recently, the use of strict anaerobic culture techniques and molecular technologies have identified other potential pathogens including anaerobic bacteria. Determining the role of all bacteria in a complex bacterial community and how they interact is extremely important; individual bacteria may affect how the community develops, possess virulence factors, produce quorum-sensing signals, stimulate an immune response or transfer antibiotic resistance genes, which could all contribute to disease progression. There are many challenges to managing cystic fibrosis lung infection but as knowledge about the airway microbiome continues to increase, this may lead to advances in the therapeutic management of the disease. © 2011 Future Medicine Ltd.

THE CULTURE MICROBIOME IN THE LUNGS OF PATIENTS WITH COPD

Introduction and Aims: Previous studies have shown that the lungs of Cystic Fibrosis (CF) and bronchiectasis (BE, not caused by CF) patients are colonised by a range of aerobic and anaerobic bacteria. As bacteria are also implicated in the pathogenesis and progression of chronic obstructive pulmonary disease (COPD), this study aimed to determine the culture microbiome of the COPD airways.

Methods: Samples were collected from 13 stable COPD patients during routine bronchoscopy. Bronchial washings were taken at a single location in the right middle lobe by flushing and removing 30 ml of sterile saline. Samples were cultured under strict anaerobic conditions with bacteria detected by plating on both selective and non-selective agar media and quantified by total viable count (TVC). Identification of the cultured bacteria was performed by amplification and subsequent sequencing of the 16sRNA gene.

Results: Mean FEV1 was 1.36 (range 0.84–2.26, mean per cent predicted FEV1, 54%), and the mean ratio (FEV1/FVC) was 51%. Bacteria were detected in 12/13 samples (92%) with bacteria from the genera Streptococcus [12/13 samples, 92%; mean (range) TVC 9.62×105 cfu/ml (1.50×103–1.42×107)] and Haemophilus [4/13 samples, 31%; mean (range) 6.40×104 cfu/ml (2.20×103–1.60×105)] most frequently detected. Anaerobic bacteria primarily from the genera Prevotella [8/13 samples, 62%; mean (range) TVC 1.12×104 cfu/ml (1.30×103–4.20×104)] and Veillonella [5/13 samples, 38%; mean (range) TVC 1.29×105 cfu/ml (4.20×103–3.60×105)] were also detected. Pseudomonas and Moraxella were not detected in any samples.

Conclusions: Our results show that bacteria from the genera Streptococcus, Haemophilus, Prevotella and Veillonella are frequently present the airways of patients suffering from COPD. Taking account of the dilutional effect of the bronchial wash procedure and extrapolating to allow comparison with sputum data in our laboratory for CF and BE, the relative load of bacteria from the genera Streptococcus, Prevotella and Veillonella is similar in these three airway diseases. The potential role of these bacteria in the progression and pathogenesis of COPD requires further investigation.

Antibiotic Management of Lung Infections in Cystic Fibrosis. I. The Microbiome, Methicillin-Resistant Staphylococcus aureus, Gram-Negative Bacteria, and Multiple Infections

Despite significant advances in treatment strategies targeting the underlying defect in cystic fibrosis (CF), airway infection remains an important cause of lung disease. In this two-part series, we review recent evidence related to the complexity of CF airway infection, explore data suggesting the relevance of individual microbial species, and discuss current and future treatment options. In Part I, the evidence with respect to the spectrum of bacteria present in the CF airway, known as the lung microbiome is discussed. Subsequently, the current approach to treat methicillin-resistant Staphylococcus aureus, gram-negative bacteria, as well as multiple coinfections is reviewed. Newer molecular techniques have demonstrated that the airway microbiome consists of a large number of microbes, and the balance between microbes, rather than the mere presence of a single species, may be relevant for disease pathophysiology. A better understanding of this complex environment could help define optimal treatment regimens that target pathogens without affecting others. Although relevance of these organisms is unclear, the pathologic consequences of methicillin-resistant S. aureus infection in patients with CF have been recently determined. New strategies for eradication and treatment of both acute and chronic infections are discussed. Pseudomonas aeruginosa plays a prominent role in CF lung disease, butmany other nonfermenting gram-negative bacteria are also found in the CF airway. Many new inhaled antibiotics specifically targeting P. aeruginosa have become available with the hope that they will improve the quality of life for patients. Part I concludes with a discussion of how best to treat patients with multiple coinfections.

Ecological and biotechnological potential of sponge microbiome

Marine sponges harbor microbial communities of immense ecological and biotechnological importance. Recently, they have been focus of heightened attention due to the wide range of biologically active compounds with potential application, particularly, in chemical, cosmetic and pharmaceutical industries. However, we still lack fundamental knowledge of their microbial ecology and biotechnological potential. The development of high-throughput sequencing technologies has given rise to a new range of tools that can help us explore the biotechnological potential of sponges with incredible detail. Metagenomics, in particular, has the power to revolutionize the production of bioactive compounds produced by unculturable microorganisms. It can offer the identification of biosynthetic genes or gene clusters that can be heterologously expressed on a cultivable and suitable host. This review focus on the exploration of the biotechnological potential of sponge-associated microorganisms, and integration of molecular approaches, whose increasing efficiency can play an essential role on achieving a sustainable source of natural products.

Metagenomic systems biology: frameworks for modeling and characterizing the gut microbiome

Thesis (Ph.D.)--University of Washington, 2014

Akkermansia muciniphila and improved metabolic health during a dietary intervention in obesity: relationship with gut microbiome richness and ecology

Objective: Individuals with obesity and type 2 diabetes differ from lean and healthy individuals in their abundance of certain gut microbial species and microbial gene richness. Abundance of Akkermansia muciniphila, a mucin-degrading bacterium, has been inversely associated with bodyfat mass and glucose intolerance in mice, but more evidence is needed in humans. The impact of diet and weight loss on this bacterial species is unknown. Our objective was to evaluate the association between fecal A. muciniphila abundance, fecal microbiome gene richness, diet, host characteristics, and their changes after calorie restriction (CR). Design: The intervention consisted of a 6-week CR period followed by a 6-week weight stabilization (WS) diet in overweight and obese adults (N=49, including 41 women). Fecal A. muciniphila abundance, fecal microbial gene richness, diet and bioclinical parameters were measured at baseline and after CR and WS. Results: At baseline A. muciniphila was inversely related to fasting glucose, waist-to-hip ratio, and subcutaneous adipocyte diameter. Subjects with higher gene richness and A. muciniphila abundance exhibited the healthiest metabolic status, particularly in fasting plasma glucose, plasma triglycerides and body fat distribution. Individuals with higher baseline A. muciniphila displayed greater improvement in insulin sensitivity markers and other clinical parameters after CR. A. muciniphila was associated with microbial species known to be related to health. Conclusion: A. muciniphila is associated with a healthier metabolic status and better clinicaloutcomes after CR in overweight/obese adults, however the interaction between gut microbiota ecology and A. muciniphila has to be taken into account.

Quantifying diet-induced metabolic changes of the human gut microbiome

The human gut microbiome is known to be associated with various human disorders, but a major challenge is to go beyond association studies and elucidate causalities. Mathematical modeling of the human gut microbiome at a genome scale is a useful tool to decipher microbe-microbe, diet-microbe and microbe-host interactions. Here, we describe the CASINO (Community And Systems-level INteractive Optimization) toolbox, a comprehensive computational platform for analysis of microbial communities through metabolic modeling. We first validated the toolbox by simulating and testing the performance of single bacteria and whole communities in vitro. Focusing on metabolic interactions between the diet, gut microbiota, and host metabolism, we demonstrated the predictive power of the toolbox in a diet-intervention study of 45 obese and overweight individuals and validated our predictions by fecal and blood metabolomics data. Thus, modeling could quantitatively describe altered fecal and serum amino acid levels in response to diet intervention.