Exploiting lung adaptation and phage steering to clear pan-resistant Pseudomonas aeruginosa infections in vivo.

Pseudomonas aeruginosa is a major nosocomial pathogen that causes severe disease including sepsis. Carbapenem-resistant P. aeruginosa is recognised by the World Health Organisation as a priority 1 pathogen, with urgent need for new therapeutics. As such, there is renewed interest in using bacteriophages as a therapeutic. However, the dynamics of treating pan-resistant P. aeruginosa with phage in vivo are poorly understood. Using a pan-resistant P. aeruginosa in vivo infection model, phage therapy displays strong therapeutic potential, clearing infection from the blood, kidneys, and spleen. Remaining bacteria in the lungs and liver displays phage resistance due to limiting phage adsorption. Yet, resistance to phage results in re-sensitisation to a wide range of antibiotics. In this work, we use phage steering in vivo, pre-exposing a pan resistant P. aeruginosa infection with a phage cocktail to re-sensitise bacteria to antibiotics, clearing the infection from all organs.

Correction: Plasmids manipulate bacterial behaviour through translational regulatory crosstalk.

[This corrects the article DOI: 10.1371/journal.pbio.3001988.].

IMG/PR: a database of plasmids from genomes and metagenomes with rich annotations and metadata.

Plasmids are mobile genetic elements found in many clades of Archaea and Bacteria. They drive horizontal gene transfer, impacting ecological and evolutionary processes within microbial communities, and hold substantial importance in human health and biotechnology. To support plasmid research and provide scientists with data of an unprecedented diversity of plasmid sequences, we introduce the IMG/PR database, a new resource encompassing 699 973 plasmid sequences derived from genomes, metagenomes and metatranscriptomes. IMG/PR is the first database to provide data of plasmid that were systematically identified from diverse microbiome samples. IMG/PR plasmids are associated with rich metadata that includes geographical and ecosystem information, host taxonomy, similarity to other plasmids, functional annotation, presence of genes involved in conjugation and antibiotic resistance. The database offers diverse methods for exploring its extensive plasmid collection, enabling users to navigate plasmids through metadata-centric queries, plasmid comparisons and BLAST searches. The web interface for IMG/PR is accessible at https://img.jgi.doe.gov/pr. Plasmid metadata and sequences can be downloaded from https://genome.jgi.doe.gov/portal/IMG_PR.

Implementation of anaerobic digestion for valorizing the organic fraction of municipal solid waste in developing countries: Technical insights from a systematic review.

Anaerobic digestion (AD) as a waste management strategy for the organic fraction of municipal waste (OFMSW) has received attention in developed countries for several decades, leading to the development of large-scale plants. In contrast, AD of OFMSW has only recently drawn attention in developing countries. This systematic review was carried out to investigate the implementation of AD to treat the OFMSW in developing countries, focusing on assessing pilot and full-scale AD plants reported in the last ten years. Studies that met the selection criteria were analyzed and data regarding operating parameters, feedstock characteristics, and biogas, digestate, and energy production were extracted. As outlined in this systematic review, AD plants located in developing countries are mostly one-stage mesophilic systems that treat OFMSW via mono-digestion, almost exclusively with the aim of producing electrical energy. Based on the analysis done throughout this systematic review, it was noted that there is a large difference in the maturity level of AD systems between developing and developed countries, mainly due to the economic capacity of developed countries to invest in sustainable waste management systems. However, the number of AD plants reported in scientific papers is significantly lower than the number of installed AD systems. Research articles regarding large-scale implementation of AD to treat OFMSW in developed countries were analyzed and compared with developing countries. This comparison identified practices used in plants in developed countries that could be utilized in the large-scale implementation and success of AD in developing countries. These practices include exploiting potential products with high market-values, forming partnerships with local industries to use industrial wastes as co-substrates, and exploring different biological and physical pretreatment technologies. Additionally, the analysis of capital and operational costs of AD plants showed that costs tend to be higher for developing countries due to their need to import of materials and equipment from developed countries. Technical, economical, and political challenges for the implementation of AD at a large-scale in developing countries are highlighted.

Emergent Lag Phase in Flux-Regulation Models of Bacterial Growth.

Lag phase is observed in bacterial growth during a sudden change in conditions: growth is inhibited whilst cells adapt to the environment. Bi-phasic, or diauxic growth is commonly exhibited by many species. In the presence of two sugars, cells initially grow by consuming the preferred sugar then undergo a lag phase before resuming growth on the second. Biomass increase is characterised by a diauxic growth curve: exponential growth followed by a period of no growth before a second exponential growth. Recent literature lacks a complete dynamic description, artificially modelling lag phase and employing non-physical representations of precursor pools. Here, we formulate a rational mechanistic model based on flux-regulation/proteome partitioning with a finite precursor pool that reveals core mechanisms in a compact form. Unlike earlier systems, the characteristic dynamics emerge as part of the solution, including the lag phase. Focussing on growth of Escherichia coli on a glucose-lactose mixture we show results accurately reproduce experiments. We show that for a single strain of E. coli, diauxic growth leads to optimised biomass yields. However, intriguingly, for two competing strains diauxic growth is not always the best strategy. Our description can be generalised to model multiple different microorganisms and investigate competition between species/strains.

Compensatory mutations reducing the fitness cost of plasmid carriage occur in plant rhizosphere communities.

Plasmids drive bacterial evolutionary innovation by transferring ecologically important functions between lineages, but acquiring a plasmid often comes at a fitness cost to the host cell. Compensatory mutations, which ameliorate the cost of plasmid carriage, promote plasmid maintenance in simplified laboratory media across diverse plasmid-host associations. Whether such compensatory evolution can occur in more complex communities inhabiting natural environmental niches where evolutionary paths may be more constrained is, however, unclear. Here, we show a substantial fitness cost of carrying the large conjugative plasmid pQBR103 in Pseudomonas fluorescens SBW25 in the plant rhizosphere. This plasmid fitness cost could be ameliorated by compensatory mutations affecting the chromosomal global regulatory system gacA/gacS, which arose rapidly in plant rhizosphere communities and were exclusive to plasmid carriers. These findings expand our understanding of the importance of compensatory evolution in plasmid dynamics beyond simplified lab media. Compensatory mutations contribute to plasmid survival in bacterial populations living within complex microbial communities in their environmental niche.

Plasmids manipulate bacterial behaviour through translational regulatory crosstalk.

Beyond their role in horizontal gene transfer, conjugative plasmids commonly encode homologues of bacterial regulators. Known plasmid regulator homologues have highly targeted effects upon the transcription of specific bacterial traits. Here, we characterise a plasmid translational regulator, RsmQ, capable of taking global regulatory control in Pseudomonas fluorescens and causing a behavioural switch from motile to sessile lifestyle. RsmQ acts as a global regulator, controlling the host proteome through direct interaction with host mRNAs and interference with the host's translational regulatory network. This mRNA interference leads to large-scale proteomic changes in metabolic genes, key regulators, and genes involved in chemotaxis, thus controlling bacterial metabolism and motility. Moreover, comparative analyses found RsmQ to be encoded on a large number of divergent plasmids isolated from multiple bacterial host taxa, suggesting the widespread importance of RsmQ for manipulating bacterial behaviour across clinical, environmental, and agricultural niches. RsmQ is a widespread plasmid global translational regulator primarily evolved for host chromosomal control to manipulate bacterial behaviour and lifestyle.

Benthic Dissolved Silicon and Iron Cycling at Glaciated Patagonian Fjord Heads.

Glacier meltwater supplies silicon (Si) and iron (Fe) sourced from weathered bedrock to downstream ecosystems. However, the extent to which these nutrients reach the ocean is regulated by the nature of the benthic cycling of dissolved Si and Fe within fjord systems, given the rapid deposition of reactive particulate fractions at fjord heads. Here, we examine the benthic cycling of the two nutrients at four Patagonian fjord heads through geochemical analyses of sediment pore waters, including Si and Fe isotopes (δ30Si and δ56Fe), and reaction-transport modeling for Si. A high diffusive flux of dissolved Fe from the fjord sediments (up to 0.02 mmol m-2 day-1) compared to open ocean sediments (typically <0.001 mmol m-2 day-1) is supported by both reductive and non-reductive dissolution of glacially-sourced reactive Fe phases, as reflected by the range of pore water δ56Fe (-2.7 to +0.8‰). In contrast, the diffusive flux of dissolved Si from the fjord sediments (0.02-0.05 mmol m-2 day-1) is relatively low (typical ocean values are >0.1 mmol m-2 day-1). High pore water δ30Si (up to +3.3‰) observed near the Fe(II)-Fe(III) redox boundary is likely associated with the removal of dissolved Si by Fe(III) mineral phases, which, together with high sedimentation rates, contribute to the low diffusive flux of Si at the sampled sites. Our results suggest that early diagenesis promotes the release of dissolved Fe, yet suppresses the release of dissolved Si at glaciated fjord heads, which has significant implications for understanding the downstream transport of these nutrients along fjord systems.

Horizontal gene transfer and ecological interactions jointly control microbiome stability.

Genes encoding resistance to stressors, such as antibiotics or environmental pollutants, are widespread across microbiomes, often encoded on mobile genetic elements. Yet, despite their prevalence, the impact of resistance genes and their mobility upon the dynamics of microbial communities remains largely unknown. Here we develop eco-evolutionary theory to explore how resistance genes alter the stability of diverse microbiomes in response to stressors. We show that adding resistance genes to a microbiome typically increases its overall stability, particularly for genes on mobile genetic elements with high transfer rates that efficiently spread resistance throughout the community. However, the impact of resistance genes upon the stability of individual taxa varies dramatically depending upon the identity of individual taxa, the mobility of the resistance gene, and the network of ecological interactions within the community. Nonmobile resistance genes can benefit susceptible taxa in cooperative communities yet damage those in competitive communities. Moreover, while the transfer of mobile resistance genes generally increases the stability of previously susceptible recipient taxa to perturbation, it can decrease the stability of the originally resistant donor taxon. We confirmed key theoretical predictions experimentally using competitive soil microcosm communities. Here the stability of a susceptible microbial community to perturbation was increased by adding mobile resistance genes encoded on conjugative plasmids but was decreased when these same genes were encoded on the chromosome. Together, these findings highlight the importance of the interplay between ecological interactions and horizontal gene transfer in driving the eco-evolutionary dynamics of diverse microbiomes.

A tale of two plasmids: contributions of plasmid associated phenotypes to epidemiological success among Shigella.

Dissemination of antimicrobial resistance (AMR) genes by horizontal gene transfer (HGT) mediated through plasmids is a major global concern. Genomic epidemiology studies have shown varying success of different AMR plasmids during outbreaks, but the underlying reasons for these differences are unclear. Here, we investigated two Shigella plasmids (pKSR100 and pAPR100) that circulated in the same transmission network but had starkly contrasting epidemiological outcomes to identify plasmid features that may have contributed to the differences. We used plasmid comparative genomics to reveal divergence between the two plasmids in genes encoding AMR, SOS response alleviation and conjugation. Experimental analyses revealed that these genomic differences corresponded with reduced conjugation efficiencies for the epidemiologically successful pKSR100, but more extensive AMR, reduced fitness costs, and a reduced SOS response in the presence of antimicrobials, compared with the less successful pAPR100. The discrepant phenotypes between the two plasmids are consistent with the hypothesis that plasmid-associated phenotypes contribute to determining the epidemiological outcome of AMR HGT and suggest that phenotypes relevant in responding to antimicrobial pressure and fitness impact may be more important than those around conjugation in this setting. Plasmid phenotypes could thus be valuable tools in conjunction with genomic epidemiology for predicting AMR dissemination.

Case Report: The effect of intravenous and oral antibiotics on the gut microbiome and breath volatile organic compounds over one year.

BACKGROUND: Antimicrobial resistance (AMR) is a global concern and better understanding of the gut microbiome, a known 'amplifier' of AMR, may allow future clinicians to tailor therapy to minimise this risk and offer a personalised medicine approach. To examine the gut microbiome, patients are required to provide faecal samples; more convenient and cheaper solutions need to be found. METHODS: As part of a pilot study looking at how routes of administration affect the gut microbiome in NHS patients undergoing routine clinical management for infections, we hypothesised that effects on the gut microbiome varied with the route and metabolism of antibiotic used, and these changes may be reflected in breath metabolites. We present a case report of a patient with an unusual clinical history, alongside breath metabolite and gut microbiome data taken before, during and after antibiotic therapy over a period of one year. RESULTS: We noted a shift in the dominant Bacteroides strain in the patient's gut microbiome between pre- and post-therapy samples, along with an alteration in the composition of breath metabolites. CONCLUSIONS: This study provides a framework for similar future work and highlights the need for further research on the relationships between changes in microbial gut communities and antimicrobial exposure, patient clinical status, and the metabolites of human breath.

Control of sheep flystrike: what's been tried in the past and where to from here.

Flystrike remains a serious financial and animal welfare issue for the sheep industry in Australia despite many years of research into control methods. The present paper provides an extensive review of past research on flystrike, and highlights areas that hold promise for providing long-term control options. We describe areas where the application of modern scientific advances may provide increased impetus to some novel, as well as some previously explored, control methods. We provide recommendations for research activities: insecticide resistance management, novel delivery methods for therapeutics, improved breeding indices for flystrike-related traits, mechanism of nematode-induced scouring in mature animals. We also identify areas where advances can be made in flystrike control through the greater adoption of well-recognised existing management approaches: optimal insecticide-use patterns, increased use of flystrike-related Australian Sheep Breeding Values, and management practices to prevent scouring in young sheep. We indicate that breeding efforts should be primarily focussed on the adoption and improvement of currently available breeding tools and towards the future integration of genomic selection methods. We describe factors that will impact on the ongoing availability of insecticides for flystrike control and on the feasibility of vaccination. We also describe areas where the blowfly genome may be useful in providing impetus to some flystrike control strategies, such as area-wide approaches that seek to directly suppress or eradicate sheep blowfly populations. However, we also highlight the fact that commercial and feasibility considerations will act to temper the potential for the genome to act as the basis for providing some control options.

What makes a megaplasmid?

Naturally occurring plasmids come in different sizes. The smallest are less than a kilobase of DNA, while the largest can be over three orders of magnitude larger. Historically, research has tended to focus on smaller plasmids that are usually easier to isolate, manipulate and sequence, but with improved genome assemblies made possible by long-read sequencing, there is increased appreciation that very large plasmids-known as megaplasmids-are widespread, diverse, complex, and often encode key traits in the biology of their host microorganisms. Why are megaplasmids so big? What other features come with large plasmid size that could affect bacterial ecology and evolution? Are megaplasmids 'just' big plasmids, or do they have distinct characteristics? In this perspective, we reflect on the distribution, diversity, biology, and gene content of megaplasmids, providing an overview to these large, yet often overlooked, mobile genetic elements. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.

The challenges of monitoring and manipulating anaerobic microbial communities.

Mixed anaerobic microbial communities are a key component in valorization of waste biomass via anaerobic digestion. Similar microbial communities are important as soil and animal microbiomes and have played a critical role in shaping the planet as it is today. Understanding how individual species within communities interact with others and their environment is important for improving performance and potential applications of an inherently green technology. Here, the challenges associated with making measurements critical to assessing the status of anaerobic microbial communities are considered. How these measurements could be incorporated into control philosophies and augment the potential of anaerobic microbial communities to produce different and higher value products from waste materials are discussed. The benefits and pitfalls of current genetic and molecular approaches to measuring and manipulating anaerobic microbial communities and the challenges which should be addressed to realise the potential of this exciting technology are explored.

Plasmid fitness costs are caused by specific genetic conflicts enabling resolution by compensatory mutation.

Plasmids play an important role in bacterial genome evolution by transferring genes between lineages. Fitness costs associated with plasmid carriage are expected to be a barrier to gene exchange, but the causes of plasmid fitness costs are poorly understood. Single compensatory mutations are often sufficient to completely ameliorate plasmid fitness costs, suggesting that such costs are caused by specific genetic conflicts rather than generic properties of plasmids, such as their size, metabolic burden, or gene expression level. By combining the results of experimental evolution with genetics and transcriptomics, we show here that fitness costs of 2 divergent large plasmids in Pseudomonas fluorescens are caused by inducing maladaptive expression of a chromosomal tailocin toxin operon. Mutations in single genes unrelated to the toxin operon, and located on either the chromosome or the plasmid, ameliorated the disruption associated with plasmid carriage. We identify one of these compensatory loci, the chromosomal gene PFLU4242, as the key mediator of the fitness costs of both plasmids, with the other compensatory loci either reducing expression of this gene or mitigating its deleterious effects by up-regulating a putative plasmid-borne ParAB operon. The chromosomal mobile genetic element Tn6291, which uses plasmids for transmission, remained up-regulated even in compensated strains, suggesting that mobile genetic elements communicate through pathways independent of general physiological disruption. Plasmid fitness costs caused by specific genetic conflicts are unlikely to act as a long-term barrier to horizontal gene transfer (HGT) due to their propensity for amelioration by single compensatory mutations, helping to explain why plasmids are so common in bacterial genomes.

Dynamic changes in anaerobic digester metabolic pathways and microbial populations during acclimatisation to increasing ammonium concentrations.

Transitions in microbial community structure in response to increasing ammonia concentrations were determined by monitoring mesophilic anaerobic digesters seeded with a predominantly acetoclastic methanogenic community from a sewage sludge digester. Ammonia concentration was raised by switching the feed to source segregated domestic food waste and applying two organic loading rates (OLR) and hydraulic retention times (HRT) in paired digesters. One of each pair was dosed with trace elements (TE) known to be essential to the transition, with the other unsupplemented digester acting as a control. Samples taken during the trial were used to determine the metabolic pathway to methanogenesis using 14C labelled acetate. Partitioning of 14C between the product gases was interpreted via an equation to indicate the proportion produced by acetoclastic and hydrogenotrophic routes. Archaeal and selected bacterial groups were identified by 16S rRNA sequencing, to determine relative abundance and diversity. Acclimatisation for digesters with TE was relatively smooth, but OLR and HRT influenced both metabolic route and community structure. The 14C ratio could be used quantitatively and, when interpreted alongside archaeal community structure, showed that at longer HRT and lower loading Methanobacteriaceae were dominant and hydrogenotrophic activity accounted for 77% of methane production. At the higher OLR and shorter HRT, Methanosarcinaceae were dominant with the 14C ratio indicating simultaneous production of methane by acetoclastic and hydrogenotrophic pathways: the first reported observation of this in digestion under mesophilic conditions. Digesters without TE supplementation showed similar initial changes but, as expected failed to complete the transition to stable operation.

The dilution effect limits plasmid horizontal transmission in multispecies bacterial communities.

By transferring ecologically important traits between species, plasmids drive genomic divergence and evolutionary innovation in their bacterial hosts. Bacterial communities are often diverse and contain multiple coexisting plasmids, but the dynamics of plasmids in multi-species communities are poorly understood. Here, we show, using experimental multi-species communities containing two plasmids, that bacterial diversity limits the horizontal transmission of plasmids due to the 'dilution effect'; this is an epidemiological phenomenon whereby living alongside less proficient host species reduces the expected infection risk for a focal host species. In addition, plasmid horizontal transmission was also affected by plasmid diversity, such that the rate of plasmid conjugation was reduced from co-infected host cells carrying both plasmids. In diverse microbial communities, plasmid spread may be limited by the dilution effect and plasmid-plasmid interactions, reducing the rate of horizontal transmission.

Positive Selection Inhibits Plasmid Coexistence in Bacterial Genomes.

Plasmids play an important role in bacterial evolution by transferring niche-adaptive functional genes between lineages, thus driving genomic diversification. Bacterial genomes commonly contain multiple, coexisting plasmid replicons, which could fuel adaptation by increasing the range of gene functions available to selection and allowing their recombination. However, plasmid coexistence is difficult to explain because the acquisition of plasmids typically incurs high fitness costs for the host cell. Here, we show that plasmid coexistence was stably maintained without positive selection for plasmid-borne gene functions and was associated with compensatory evolution to reduce fitness costs. In contrast, with positive selection, plasmid coexistence was unstable despite compensatory evolution. Positive selection discriminated between differential fitness benefits of functionally redundant plasmid replicons, retaining only the more beneficial plasmid. These data suggest that while the efficiency of negative selection against plasmid fitness costs declines over time due to compensatory evolution, positive selection to maximize plasmid-derived fitness benefits remains efficient. Our findings help to explain the forces structuring bacterial genomes: coexistence of multiple plasmids in a genome is likely to require either rare positive selection in nature or nonredundancy of accessory gene functions among the coexisting plasmids.IMPORTANCE Bacterial genomes often contain multiple coexisting plasmids that provide important functions like antibiotic resistance. Using lab experiments, we show that such plasmid coexistence within a genome is stable only in environments where the function they encode is useless but is unstable if the function is useful and beneficial for bacterial fitness. Where competing plasmids perform the same useful function, only the most beneficial plasmid is kept by the cell, a process that is similar to competitive exclusion in ecological communities. This process helps explain how bacterial genomes are structured: bacterial genomes expand in size by acquiring multiple plasmids when selection is relaxed but subsequently contract during periods of strong selection for the useful plasmid-encoded function.