13 research outputs found

    Recovering Greater Fungal Diversity from Pristine and Diesel Fuel Contaminated Sub-Antarctic Soil Through Cultivation Using Both a High and a Low Nutrient Media Approach

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    Novel cultivation strategies for bacteria are widespread and well described for recovering greater diversity from the “hitherto” unculturable majority. While similar approaches have not yet been demonstrated for fungi it has been suggested that of the 1.5 million estimated species less than 5% have been recovered into pure culture. Fungi are known to be involved in many degradative processes, including the breakdown of petroleum hydrocarbons, and it has been speculated that in Polar Regions they contribute significantly to bioremediation of contaminated soils. Given the biotechnological potential of fungi there is a need to increase efforts for greater species recovery, particularly from extreme environments such as sub-Antarctic Macquarie Island. In this study, like the yet-to-be cultured bacteria, high concentrations of nutrients selected for predominantly different fungal species to that recovered using a low nutrient media. By combining both media approaches to the cultivation of fungi from contaminated and non-contaminated soils, 91 fungal species were recovered, including 63 unidentified species. A preliminary biodegradation activity assay on a selection of isolates found that a high proportion of novel and described fungal species from a range of soil samples were capable of hydrocarbon degradation and should be characterized further

    Microfluidic qPCR Enables High Throughput Quantification of Microbial Functional Genes but Requires Strict Curation of Primers

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    Quantification of microbial functional genes enhances predictions of soil biogeochemical process rates, but reliance on low-throughput quantitative PCR (qPCR) limits the scope of ecological studies to a handful of targets. Here, we explore whether microfluidic qPCR (MFQPCR) is a viable high-throughput alternative for functional gene quantification, by evaluating the efficiency, specificity and sensitivity of 29 established and 12 newly designed primer pairs targeting taxonomic, nitrogen-cycling, and hydrocarbon degradation genes in genomic DNA soil extracts, under three different sets of MFQPCR assay conditions. Without curation, commonly-used qPCR primer pairs yielded an extreme range of reaction efficiencies (25.9–100.1%), but when conditions were optimized, MFQPCR produced copy-number estimates comparable to traditional qPCR. To guide microbial soil ecologists considering adoption of MFQPCR, we present suggestions for primer selection, including omission of inosines, degeneracy scores of < 9, amplicon sizes of ≤ 211 bp, and GC content of 32–61%. We conclude that, while the nanoliter reaction volumes, rapid thermocycling and one-size-fits-all reaction conditions of MFQPCR necessitates more stringent primer selection criteria than is commonly applied in soil microbial ecology, the ability to quantify up to 96 targets in 96 samples makes MFQPCR a valuable tool for monitoring shifts in functional community abundances. MFQPCR will particularly suit studies targeting multiple clade-specific functional genes, or when primer design is informed by previous knowledge of the environment

    What Do We Know about the Microbiome in Cystic Fibrosis? Is There a Role for Probiotics and Prebiotics?

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    Cystic fibrosis (CF) is a life-shortening genetic disorder that affects the cystic fibrosis transmembrane conductance regulator (CFTR) protein. In the gastrointestinal (GI) tract, CFTR dysfunction results in low intestinal pH, thick and inspissated mucus, a lack of endogenous pancreatic enzymes, and reduced motility. These mechanisms, combined with antibiotic therapies, drive GI inflammation and significant alteration of the GI microbiota (dysbiosis). Dysbiosis and inflammation are key factors in systemic inflammation and GI complications including malignancy. The following review examines the potential for probiotic and prebiotic therapies to provide clinical benefits through modulation of the microbiome. Evidence from randomised control trials suggest probiotics are likely to improve GI inflammation and reduce the incidence of CF pulmonary exacerbations. However, the highly variable, low-quality data is a barrier to the implementation of probiotics into routine CF care. Epidemiological studies and clinical trials support the potential of dietary fibre and prebiotic supplements to beneficially modulate the microbiome in gastrointestinal conditions. To date, limited evidence is available on their safety and efficacy in CF. Variable responses to probiotics and prebiotics highlight the need for personalised approaches that consider an individual’s underlying microbiota, diet, and existing medications against the backdrop of the complex nutritional needs in CF

    What Do We Know about the Microbiome in Cystic Fibrosis? Is There a Role for Probiotics and Prebiotics?

    No full text
    Cystic fibrosis (CF) is a life-shortening genetic disorder that affects the cystic fibrosis transmembrane conductance regulator (CFTR) protein. In the gastrointestinal (GI) tract, CFTR dysfunction results in low intestinal pH, thick and inspissated mucus, a lack of endogenous pancreatic enzymes, and reduced motility. These mechanisms, combined with antibiotic therapies, drive GI inflammation and significant alteration of the GI microbiota (dysbiosis). Dysbiosis and inflammation are key factors in systemic inflammation and GI complications including malignancy. The following review examines the potential for probiotic and prebiotic therapies to provide clinical benefits through modulation of the microbiome. Evidence from randomised control trials suggest probiotics are likely to improve GI inflammation and reduce the incidence of CF pulmonary exacerbations. However, the highly variable, low-quality data is a barrier to the implementation of probiotics into routine CF care. Epidemiological studies and clinical trials support the potential of dietary fibre and prebiotic supplements to beneficially modulate the microbiome in gastrointestinal conditions. To date, limited evidence is available on their safety and efficacy in CF. Variable responses to probiotics and prebiotics highlight the need for personalised approaches that consider an individual’s underlying microbiota, diet, and existing medications against the backdrop of the complex nutritional needs in CF

    Microbial indicators of diesel fuel toxicity in polar soils

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    Petroleum hydrocarbon contamination is extensive throughout the Polar Regions and particularly prevalent around areas of concentrated anthropogenic activity. Whilst toxicity information is limited there is evidence to suggest that petroleum hydrocarbon contamination is more damaging near the poles than in temperate regions due to the low temperatures, low nutrient availability and extended exposure to the contaminants. As the dominant biota present in Antarctic soils, microbial populations are ideal candidates for the development of site-specific ecotoxicology assays. Bacteria in particular are valuable indicators of ecosystem health due to their pivotal role in biogeochemical cycles, nutrient cycling and ecosystem sustainability. For this thesis I have examined the response of the indigenous soil microbial community to diesel fuel toxicity at the sub-Antarctic Macquarie Island. I have employed a range of techniques to characterise the community including novel culturing, community fingerprinting, pyrosequencing and qPCR methods. A range of broad community indices and specific functional portions of the community were targeted to establish the most promising microbial indicators of diesel fuel toxicity. Similar approaches were then applied to three chronically contaminated sites under active remediation to monitor the microbial community recovery. The diversity, species richness, species evenness and similarity indices declined with increasing diesel fuel concentrations. Yet, the greatest response of the diesel fuel was to alter relative abundances of key lineages rather than remove entire species. This response was consistent across all soils but varied in the severity of the response between locations. Key processors within the nitrogen cycle were also inhibited but again the severity of the response was site-specific, highlighting the need for site-specific remediation targets to be established

    Flow cytometry in environmental microbiology : a rapid approach for the isolation of single cells for advanced molecular biology analysis

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    The isolation and subsequent characterization of microbial cells from within environmental samples is a difficult process. Flow cytometry and cell sorting, when combined with the application of fluorescent probes, have the capability for the detection and separation of diverse microbial populations from within complex mixtures. The isolation of single cells allows for downstream investigations towards system-level characterization of unknown Bacterial Phyla to occur. We describe here the combination of fluorescent in situ hybridization and cell sorting for the detection and isolation of Candidate Division TM7 bacteria from an enriched soil sample. The result is the isolation of rare cells suitable for advanced molecular analysis including whole genome amplification and high-throughput pyrosequencing.24 page(s

    Intestinal Inflammation and Alterations in the Gut Microbiota in Cystic Fibrosis: A Review of the Current Evidence, Pathophysiology and Future Directions

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    Cystic fibrosis (CF) is a life-limiting autosomal recessive multisystem disease. While its burden of morbidity and mortality is classically associated with pulmonary disease, CF also profoundly affects the gastrointestinal (GI) tract. Chronic low-grade inflammation and alterations to the gut microbiota are hallmarks of the CF intestine. The etiology of these manifestations is likely multifactorial, resulting from cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction, a high-fat CF diet, and the use of antibiotics. There may also be a bidirectional pathophysiological link between intestinal inflammation and changes to the gut microbiome. Additionally, a growing body of evidence suggests that these GI manifestations may have significant clinical associations with growth and nutrition, quality of life, and respiratory function in CF. As such, the potential utility of GI therapies and long-term GI outcomes are areas of interest in CF. Further research involving microbial modulation and multi-omics techniques may reveal novel insights. This article provides an overview of the current evidence, pathophysiology, and future research and therapeutic considerations pertaining to intestinal inflammation and alterations in the gut microbiota in CF

    Diet and the gut-lung axis in cystic fibrosis – direct & indirect links

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    ABSTRACTCystic fibrosis (CF) is a multisystem, autosomal, recessive disease primarily affecting the lungs, pancreas, gastrointestinal tract, and liver. Whilst there is increasing evidence of a microbial ‘gut-lung axis’ in chronic respiratory conditions, there has been limited analysis of such a concept in CF. We performed a comprehensive dietary and microbiota analysis to explore the interactions between diet, gastrointestinal microbiota, respiratory microbiota, and clinical outcomes in children with CF. Our results demonstrate significant alterations in intestinal inflammation and respiratory and gastrointestinal microbiota when compared to age and gender matched children without CF. We identified correlations between the gastrointestinal and respiratory microbiota, lung function, CF pulmonary exacerbations and anthropometrics, supporting the concept of an altered gut-lung axis in children with CF. We also identified significant differences in dietary quality with CF children consuming greater relative proportions of total, saturated and trans fats, and less relative proportions of carbohydrates, wholegrains, fiber, insoluble fiber, starch, and resistant starch. Our findings position the CF diet as a potential modulator in gastrointestinal inflammation and the proposed gut-lung axial relationship in CF. The dietary intake of wholegrains, fiber and resistant starch may be protective against intestinal inflammation and should be explored as potential therapeutic adjuvants for children with CF
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