ThermoPhyl : a software tool for selecting phylogenetically optimized conventional and quantitative-PCR taxon-targeted assays for use with complex samples

The ability to specifically and sensitively target genotypes of interest is critical for the success of many PCR-based analyses of environmental or clinical samples that contain multiple templates.Next-generation sequence data clearly show that such samples can harbour hundreds to thousands of operational taxonomic units; a richness which precludes the manual evaluation of candidate assay specificity and sensitivity using multiple sequence alignments. To solve this problem we have developed and validated a free software tool which automates the identification of PCR assays targeting specific genotypes in complex samples. ThermoPhyl uses user-defined target and non-target sequence databases to assess the phylogenetic sensitivity and specificity of thermodynamically optimised candidate assays derived from primer design software packages. ThermoPhyl takes its name from its central premise of testing Thermodynamically optimal assays for Phylogenetic specificity and sensitivity and can be used for two primer (traditional PCR) or two primers with an internal probe (e.g. TaqMan® qPCR) applications and potentially for oligonucleotide probes.Here we describe the use of ThermoPhyl for traditional PCR and qPCR assays. PCR assays selected using ThermoPhyl were validated using 454 pyrosequencing of a traditional specific PCR assay and with a set of four genotype-specific qPCR assays applied to estuarine sediment samples

Improved hydrogen gas production in microbial electrolysis cells using inexpensive recycled carbon fibre fabrics

Growing energy demands of wastewater treatment have made it vital for water companies to develop less energy intensive processes for treating wastewater if net zero emissions are to be achieved by 2050. Microbial electrolysis cells (MECs) have the potential to do this by treating water and producing renewable hydrogen gas as a product, but capital and operational costs have slowed their deployment. By using recycled carbon fibre mats, commercially viable MECs can brought closer to reality, where recycled carbon fibre anode MECs treating real wastewater (normalised ~3100 L d−1) were producing 66.77 L H2 d−1 while graphite felt anode MECs produced 3.65 L H2 d−1 per 1 m3 reactor, anodes costing £5.53 m−2 and £88.36 m−2 respectively, resulting in a total anode cost saving of 93%. This could incentivise the development of larger pilot systems, opening the door for generating greater value and a more sustainable wastewater treatment industry

Phenotypic and genotypic characteristics of small colony variants and their role in chronic infection

Small colony variant (SCV) bacteria arise spontaneously within apparently homogeneous microbial populations, largely in response to environmental stresses, such as antimicrobial treatment. They display unique phenotypic characteristics conferred in part by heritable genetic changes. Characteristically slow growing, SCVs comprise a minor proportion of the population from which they arise but persist by virtue of their inherent resilience and host adaptability. Consequently, SCVs are problematic in chronic infection, where antimicrobial treatment is administered during the acute phase of infection but fails to eradicate SCVs, which remain within the host causing recurrent or chronic infection. This review discusses some of the phenotypic and genotypic changes that enable SCVs to successfully proliferate within the host environment as potential pathogens and strategies that could ameliorate the resolution of infection where SCVs are present

Draft genome sequence of Pseudomonas aeruginosa ATCC 9027, originally isolated from an outer ear infection

Pseudomonas aeruginosa ATCC 9027 was isolated in 1943 from a case of otitis externa and is commonly employed as a quality control strain for sterility, assessment of antibiofilm agents, and in vitro study of wound infection. Here, we present the 6.34-Mb draft genome sequence and highlight some pertinent genes that are associated with virulence

Metagenomic data-mining reveals contrasting microbial populations responsible for trimethylamine formation in human gut and marine ecosystems

Existing metagenome datasets from many different environments contain untapped potential for understanding metabolic pathways and their biological impact. Our interest lies in the formation of trimethylamine (TMA), a key metabolite in both human health and climate change. Here, we focus on bacterial degradation pathways for choline, carnitine, glycine betaine and trimethylamine N-oxide (TMAO) to TMA in human gut and marine metagenomes. We found the TMAO reductase pathway was the most prevalent pathway in both environments. Proteobacteria were found to contribute the majority of the TMAO reductase pathway sequences, except in the stressed gut, where Actinobacteria dominated. Interestingly, in the human gut metagenomes, a high proportion of the Proteobacteria hits were accounted for by the genera Klebsiella and Escherichia. Furthermore Klebsiella and Escherichia harboured three of the four potential TMA-production pathways (choline, carnitine and TMAO), suggesting they have a key role in TMA cycling in the human gut. In addition to the intensive TMAO–TMA cycling in the marine environment, our data suggest that carnitine-to-TMA transformation plays an overlooked role in aerobic marine surface waters, whereas choline-to-TMA transformation is important in anaerobic marine sediments. Our study provides new insights into the potential key microbes and metabolic pathways for TMA formation in two contrasting environments

Metabolic flexibility as a major predictor of spatial distribution in microbial communities

A better understand the ecology of microbes and their role in the global ecosystem could be achieved if traditional ecological theories can be applied to microbes. In ecology organisms are defined as specialists or generalists according to the breadth of their niche. Spatial distribution is often used as a proxy measure of niche breadth; generalists have broad niches and a wide spatial distribution and specialists a narrow niche and spatial distribution. Previous studies suggest that microbial distribution patterns are contrary to this idea; a microbial generalist genus (Desulfobulbus) has a limited spatial distribution while a specialist genus (Methanosaeta) has a cosmopolitan distribution. Therefore, we hypothesise that this counter-intuitive distribution within generalist and specialist microbial genera is a common microbial characteristic. Using molecular fingerprinting the distribution of four microbial genera, two generalists, Desulfobulbus and the methanogenic archaea Methanosarcina, and two specialists, Methanosaeta and the sulfate-reducing bacteria Desulfobacter were analysed in sediment samples from along a UK estuary. Detected genotypes of both generalist genera showed a distinct spatial distribution, significantly correlated with geographic distance between sites. Genotypes of both specialist genera showed no significant differential spatial distribution. These data support the hypothesis that the spatial distribution of specialist and generalist microbes does not match that seen with specialist and generalist large organisms. It may be that generalist microbes, while having a wider potential niche, are constrained, possibly by intrageneric competition, to exploit only a small part of that potential niche while specialists, with far fewer constraints to their niche, are more capable of filling their potential niche more effectively, perhaps by avoiding intrageneric competition. We suggest that these counter-intuitive distribution patterns may be a common feature of microbes in general and represent a distinct microbial principle in ecology, which is a real challenge if we are to develop a truly inclusive ecology

Interaction between Staphylococcus aureus and Pseudomonas aeruginosa is beneficial for colonisation and pathogenicity in a mixed-biofilm

Debate regarding the co-existence of Staphylococcus aureus and Pseudomonas aeruginosa in wounds remains contentious, with the dominant hypothesis describing a situation akin to niche partitioning, whereby both microorganisms are present but occupy distinct regions of the wound without interacting. In contrast, we hypothesised that these microorganisms do interact during early co-colonisation in a manner beneficial to both bacteria. We assessed competitive interaction between S. aureus and P. aeruginosa in biofilm cultured for 24-72 h and bacterial aggregates analogous to those observed in early (<24h) biofilm formation, and interaction with human keratinocytes. We observed that S. aureus predominated in biofilm and non-attached bacterial aggregates, acting as a pioneer for the attachment of P. aeruginosa. We report for the first time that S. aureus mediates a significant (P<0.05) increase in the attachment of P. aeruginosa to human keratinocytes, and that P. aeruginosa promotes an invasive phenotype in S. aureus. We show that co-infected keratinocytes exhibit an intermediate inflammatory response concurrent with impaired wound closure that is in keeping with a sustained pro-inflammatory response which allows for persistent microbial colonisation. These studies demonstrate that, contrary to the dominant hypothesis, interactions between S. aureus and P. aeruginosa may be an important factor for both colonisation and pathogenicity in the chronic infected wound

A rationale for higher ratios of CH4 to CO2 production in warmer anoxic freshwater sediments and soils

Freshwaters emit significant amounts of CH4 and CO2 and, as CH4 is the stronger greenhouse gas, understanding how carbon gets mineralized to either gas is important. In theory, under anoxia, methanogenesis coupled to fermentation should produce CH4 and CO2 in a 1 : 1 ratio. Here, we find that this 1 : 1 ratio is rare, with lower ratios of 0.1 : 1 being typical which confounds understanding CH4 in freshwaters. First, using a simple mathematical model we rationalize low ratios as poor methanogenic substrate utilization, including loss to nonmethanogenic processes. Second, we find substrate utilization improves at higher temperatures, especially for hydrogen. This increases CH4 to CO2 production ratios exponentially which could drive higher CH4 to CO2 emission ratios. Hence, we rationalize how warmer freshwaters may emit more methane

Differences in composition of interdigital skin microbiota predict sheep and feet that develop footrot

Footrot has a major impact on health and productivity of sheep worldwide. The current paradigm for footrot pathogenesis is that physical damage to the interdigital skin (IDS) facilitates invasion of the essential pathogen Dichelobacter nodosus. The composition of the IDS microbiota is different in healthy and diseased feet, so an alternative hypothesis is that changes in the IDS microbiota facilitate footrot. We investigated the composition and diversity of the IDS microbiota of ten sheep, five that did develop footrot and five that did not (healthy) at weekly intervals for 20 weeks. The IDS microbiota was less diverse on sheep 2 + weeks before they developed footrot than on healthy sheep. This change could be explained by only seven of > 2000 bacterial taxa detected. The incubation period of footrot is 8–10 days, and there was a further reduction in microbial diversity on feet that developed footrot in that incubation period. We conclude that there are two stages of dysbiosis in footrot: the first predisposes sheep to footrot and the second occurs in feet during the incubation of footrot. These findings represent a step change in our understanding of the role of the IDS microbiota in footrot pathogenesis

Direct biological fixation provides a freshwater sink for N2O

Nitrous oxide (N2O) is a potent climate gas, with its strong warming potential and ozone-depleting properties both focusing research on N2O sources. Although a sink for N2O through biological fixation has been observed in the Pacific, the regulation of N2O-fixation compared to canonical N2-fixation is unknown. Here we show that both N2O and N2 can be fixed by freshwater communities but with distinct seasonalities and temperature dependencies. N2O fixation appears less sensitive to temperature than N2 fixation, driving a strong sink for N2O in colder months. Moreover, by quantifying both N2O and N2 fixation we show that, rather than N2O being first reduced to N2 through denitrification, N2O fixation is direct and could explain the widely reported N2O sinks in natural waters. Analysis of the nitrogenase (nifH) community suggests that while only a subset is potentially capable of fixing N2O they maintain a strong, freshwater sink for N2O that could be eroded by warming