Optimizing bacteriophage treatment of resistant Pseudomonas.

Phage therapy is increasing in relevance as an alternative treatment to combat antibiotic resistant bacteria. Phage cocktails are the state-of-the-art method of administering phages in clinical settings, preferred over monophage treatment because of their ability to eliminate multiple bacterial strains and reduce resistance formation. In our study, we compare monophage applications and phage cocktails to our chosen method of phage sequential treatments. To do so, we isolated four novel bacteriophages capable of infecting Pseudomonas alcaligenes T3, a close relative of P. aeruginosa, and characterized them using sequencing and transmission electron microscopy. While investigating monophage treatments, we observed that different phage concentrations had a strong impact on the timing and amount of resistance formation. When using phage cocktails, we observed that P. alcaligenes were capable of forming resistance in the same timespan it took them to become resistant to single phages. We isolated mutants resistant to each single phage as well as mutants exposed to phage cocktails, resulting in bacteria resistant to all four phages at once. Sequencing these mutants showed that different treatments yielded unique single nucleotide polymorphism mutation patterns. In order to combat resistance formation, we added phages one by one in intervals of 24 h, thus managing to delay resistance development and keeping bacterial growth significantly lower compared to phage cocktails.IMPORTANCEWHO declared antimicrobial resistance a top threat to global health; while antibiotics have stood at the forefront in the fight against bacterial infection, the increasing number of multidrug-resistant bacteria highlights a need to branch out in order to address the threat of antimicrobial resistance. Bacteriophages, viruses solely infecting bacteria, could present a solution due to their abundance, versatility, and adaptability. For this study, we isolated new phages infecting a fast-mutating Pseudomonas alcaligenes strain capable of forming resistance within 30 h. By using a sequential treatment approach of adding one phage after another, we were able to curb bacterial growth significantly more compared to state-of-the-art phage cocktails.

Spatial Distribution and Functional Impact of Human Scalp Hair Follicle Microbiota.

Human hair follicles (HFs) constitute a unique microbiota habitat that differs substantially from the skin surface. Traditional HF sampling methods fail to eliminate skin microbiota contaminants or assess the HF microbiota incompletely, and microbiota functions in human HF physiology remain ill explored. Therefore, we used laser-capture microdissection, metagenomic shotgun sequencing, and FISH to characterize the human scalp HF microbiota in defined anatomical compartments. This revealed significant compartment-, tissue lineage-, and donor age-dependent variations in microbiota composition. Greatest abundance variations between HF compartments were observed for viruses, archaea, Staphylococcus epidermidis, Cutibacterium acnes, and Malassezia restricta, with the latter 2 being the most abundant viable HF colonizers (as tested by propidium monoazide assay) and, surprisingly, most abundant in the HF mesenchyme. Transfection of organ-cultured human scalp HFs with S. epidermidis-specific lytic bacteriophages ex vivo downregulated transcription of genes known to regulate HF growth and development, metabolism, and melanogenesis, suggesting that selected microbial products may modulate HF functions. Indeed, HF treatment with butyrate, a metabolite of S. epidermidis and other HF microbiota, delayed catagen and promoted autophagy, mitochondrial activity, and gp100 and dermcidin expression ex vivo. Thus, human HF microbiota show spatial variations in abundance and modulate the physiology of their host, which invites therapeutic targeting.

Multiple neuronal populations control the eating behavior in Hydra and are responsive to microbial signals.

Although recent studies indicate the impact of microbes on the central nervous systems and behavior, it remains unclear how the relationship between the functionality of the nervous system, behavior, and the microbiota evolved. In this work, we analyzed the eating behavior of Hydra, a host that has a simple nervous system and a low-complexity microbiota. To identify the neuronal subpopulations involved, we used a subpopulation-specific cell ablation system and calcium imaging. The role of the microbiota was uncovered by manipulating the diversity of the natural microbiota. We show that different neuronal subpopulations are functioning together to control eating behavior. Animals with a drastically reduced microbiome had severe difficulties in mouth opening due to a significantly increased level of glutamate. This could be reversed by adding a full complement of the microbiota. In summary, we provide a mechanistic explanation of how Hydra's nervous system controls eating behavior and what role microbes play in this.

Impact of antibiotic perturbation on fecal viral communities in mice.

Viruses and bacteriophages have a strong impact on intestinal barrier function and the composition and functional properties of commensal bacterial communities. Shifts of the fecal virome might be involved in human diseases, including inflammatory bowel disease (IBD). Loss-of-function variants in the nucleotide-binding oligomerization domain-containing protein 2 (NOD2) gene are associated with an increased risk of developing Crohn's disease, a subtype of human chronic IBD, where specific changes in fecal viral communities have also been described. To improve our understanding of the dynamics of the enteric virome, we longitudinally characterized the virome in fecal samples from wild-type C57BL/6J and NOD2 knock-out mice in response to an antibiotic perturbation. Sequencing of virus-like particles demonstrated both a high diversity and high interindividual variation of the murine fecal virome composed of eukaryotic viruses and bacteriophages. Antibiotics had a significant impact on the fecal murine virome. Viral community composition only partially recovered in the observation period (10 weeks after cessation of antibiotics) irrespective of genotype. However, compositional shifts in the virome and bacteriome were highly correlated, suggesting that the loss of specific phages may contribute to prolonged dysregulation of the bacterial community composition. We suggest that therapeutic interference with the fecal virome may represent a novel approach in microbiota-targeted therapies.

Adaptive lifestyle of bacteria determines phage-bacteria interaction.

Bacteriophages and their interactions with microbes are not well understood. As a first step toward achieving a better understanding, we isolated and sequenced the Curvibacter phage PCA1 for the purpose of eliminating Curvibacter sp. AEP1.3, the main colonizer of Hydra vulgaris AEP. Our experiments showed that PCA1 phage caused a strong, virulent infection only in sessile Curvibacter sp. AEP1.3 but was unable to infect planktonic and host-associated bacterial cells of the same strain. In an effort to investigate this phenomenon, we compared sessile, planktonic, and host-associated bacteria via RNA sequencing and found that all three states differed significantly in their expression patterns. This finding led us to propose that the adaptive lifestyle of Curvibacter sp. AEP1.3 results in varying degrees of susceptibility to bacteriophage infection. This concept could be relevant for phage research and phage therapy in particular. Finally, we were able to induce phage infection in planktonic cells and pinpoint the infection process to a membrane protein. We further identified potential phage-binding protein candidates based on expression pattern analysis.

Symbiotic Algae of Hydra viridissima Play a Key Role in Maintaining Homeostatic Bacterial Colonization.

The freshwater polyp Hydra viridissima (H. viridissima) harbors endosymbiotic Chlorella algae in addition to a species-specific microbiome. The molecular basis of the symbiosis between Hydra and Chlorella has been characterized to be metabolic in nature. Here, we studied the interaction between the extracellularly located microbiota and the algal photobiont, which resides in Hydra's endodermal epithelium, with main focus on Legionella bacterium. We aimed at evaluating the influence of the symbiotic algae on microbial colonization and in shaping the host microbiome. We report that the microbiome composition of symbiotic and aposymbiotic (algae free) H. viridissima is significantly different and dominated by Legionella spp. Hvir in aposymbiotic animals. Co-cultivation of these animals resulted in horizontal transmission of Legionella spp. Hvir bacteria from aposymbiotic to symbiotic animals. Acquisition of this bacterium increased the release of algae into ambient water. From there, algae could subsequently be taken up again by the aposymbiotic animals. The presence of algal symbionts had negative impact on Legionella spp. Hvir and resulted in a decrease of the relative abundance of this bacterium. Prolonged co-cultivation ultimately resulted in the disappearance of the Legionella spp. Hvir bacterium from the Hydra tissue. Our observations suggest an important role of the photobiont in controlling an invasive species in a metacommunity and, thereby, shaping the microbiome.

Hydra and the hair follicle - An unconventional comparative biology approach to exploring the human holobiont.

The microbiome of human hair follicles (HFs) has emerged as an important player in different HF and skin pathologies, yet awaits in-depth exploration. This raises questions regarding the tightly linked interactions between host environment, nutrient dependency of host-associated microbes, microbial metabolism, microbe-microbe interactions and host immunity. The use of simple model systems facilitates addressing generally important questions and testing overarching, therapeutically relevant principles that likely transcend obvious interspecies differences. Here, we evaluate the potential of the freshwater polyp Hydra, to dissect fundamental principles of microbiome regulation by the host, that is the human HF. In particular, we focus on therapeutically targetable host-microbiome interactions, such as nutrient dependency, microbial interactions and host defence. Offering a new lens into the study of HF - microbiota interactions, we argue that general principles of how Hydra manages its microbiota can inform the development of novel, microbiome-targeting therapeutic interventions in human skin disease.

A Phage Protein Aids Bacterial Symbionts in Eukaryote Immune Evasion.

Phages are increasingly recognized as important members of host-associated microbiomes, with a vast genomic diversity. The new frontier is to understand how phages may affect higher order processes, such as in the context of host-microbe interactions. Here, we use marine sponges as a model to investigate the interplay between phages, bacterial symbionts, and eukaryotic hosts. Using viral metagenomics, we find that sponges, although massively filtering seawater, harbor species-specific and even individually unique viral signatures that are taxonomically distinct from other environments. We further discover a symbiont phage-encoded ankyrin-domain-containing protein, which is widely spread in phages of many host-associated contexts including human. We confirm in macrophage infection assays that the ankyrin protein (ANKp) modulates the eukaryotic host immune response against bacteria. We predict that the role of ANKp in nature is to facilitate coexistence in the tripartite interplay between phages, symbionts, and sponges and possibly many other host-microbe associations.

Exposure of the Host-Associated Microbiome to Nutrient-Rich Conditions May Lead to Dysbiosis and Disease Development-an Evolutionary Perspective.

Inflammatory diseases, such as inflammatory bowel diseases, are dramatically increasing worldwide, but an understanding of the underlying factors is lacking. We here present an ecoevolutionary perspective on the emergence of inflammatory diseases. We propose that adaptation has led to fine-tuned host-microbe interactions, which are maintained by secreted host metabolites nourishing the associated microbes. A constant elevation of nutrients in the gut environment leads to an increased activity and changed functionality of the microbiota, thus severely disturbing host-microbe interactions and leading to dysbiosis and disease development. In the past, starvation and pathogen infections, causing diarrhea, were common incidences that reset the gut bacterial community to its "human-specific-baseline." However, these natural clearing mechanisms have been virtually eradicated in developed countries, allowing a constant uncontrolled growth of bacteria. This leads to an increase of bacterial products that stimulate the immune system and ultimately might initiate inflammatory reactions.

Contrasting distributions of bacteriophages and eukaryotic viruses from contaminated coastal sediments.

Viruses are ubiquitous, abundant and play an important role in all ecosystems. Here, we advance understanding of coastal sediment viruses by exploring links in the composition and abundance of sediment viromes to environmental stressors and sediment bacterial communities. We collected sediment from contaminated and reference sites in Sydney Harbour and used metagenomics to analyse viral community composition. The proportion of phages at contaminated sites was significantly greater than phages at reference sites, whereas eukaryotic viruses were relatively more abundant at reference sites. We observed shifts in viral and bacterial composition between contaminated and reference sites of a similar magnitude. Models based on sediment characteristics revealed that total organic carbon in the sediments explained most of the environmental stress-related variation in the viral dataset. Our results suggest that the presence of anthropogenic contaminants in coastal sediments could be influencing viral community composition with potential consequences for associated hosts and the environment.

Metaorganisms in extreme environments: do microbes play a role in organismal adaptation?

From protists to humans, all animals and plants are inhabited by microbial organisms. There is an increasing appreciation that these resident microbes influence the fitness of their plant and animal hosts, ultimately forming a metaorganism consisting of a uni- or multicellular host and a community of associated microorganisms. Research on host-microbe interactions has become an emerging cross-disciplinary field. In both vertebrates and invertebrates a complex microbiome confers immunological, metabolic and behavioural benefits; conversely, its disturbance can contribute to the development of disease states. However, the molecular and cellular mechanisms controlling the interactions within a metaorganism are poorly understood and many key interactions between the associated organisms remain unknown. In this perspective article, we outline some of the issues in interspecies interactions and in particular address the question of how metaorganisms react and adapt to inputs from extreme environments such as deserts, the intertidal zone, oligothrophic seas, and hydrothermal vents.

Temperate phages as self-replicating weapons in bacterial competition.

Microbial communities are accompanied by a diverse array of viruses. Through infections of abundant microbes, these viruses have the potential to mediate competition within the community, effectively weakening competitive interactions and promoting coexistence. This is of particular relevance for host-associated microbial communities, because the diversity of the microbiota has been linked to host health and functioning. Here, we study the interaction between two key members of the microbiota of the freshwater metazoan Hydra vulgaris The two commensal bacteria Curvibacter sp. and Duganella sp. protect their host from fungal infections, but only if both of them are present. Coexistence of the two bacteria is thus beneficial for Hydra Intriguingly, Duganella sp. appears to be the superior competitor in vitro due to its higher growth rate when both bacteria are grown separately, but in co-culture the outcome of competition depends on the relative initial abundances of the two species. The presence of an inducible prophage in the Curvibacter sp. genome, which is able to lytically infect Duganella sp., led us to hypothesize that the phage modulates the interaction between these two key members of the Hydra microbiota. Using a mathematical model, we show that the interplay of the lysogenic life cycle of the Curvibacter phage and the lytic life cycle on Duganella sp. can explain the observed complex competitive interaction between the two bacteria. Our results highlight the importance of taking lysogeny into account for understanding microbe-virus interactions and show the complex role phages can play in promoting coexistence of their bacterial hosts.

Competing forces maintain the Hydra metaorganism.

Our conventional view of multicellular organisms often overlooks the fact that they are metaorganisms. They consist of a host, which is comprised of both a community of self-replicating cells that can compete as well as cooperate and a community of associated microorganisms. This newly discovered complexity raises a profound challenge: How to maintain such a multicellular association that includes independently replicating units and even different genotypes? Here, we identify competing forces acting at the host tissue level, the host-microbe interface, and within the microbial community as key factors to maintain the metaorganism Hydra. Maintenance of host tissue integrity, as well as proper regulation and management of the multiorganismic interactions are fundamental to organismal survival and health. Findings derived from the in vivo context of the Hydra model may provide one of the simplest possible systems to address questions on how a metaorganism is established and remains in balance over time.

Microbial ecology in Hydra: why viruses matter.

While largely studied because of their harmful effects on human health, there is growing appreciation that viruses are also important members of the animal holobiont. This review highlights recent findings on viruses associated with Hydra and related Cnidaria. These early evolutionary diverging animals not only select their bacterial communities but also select for viral communities in a species-specific manner. The majority of the viruses associating with these animals are bacteriophages. We demonstrate that the animal host and its virome have evolved into a homeostatic, symbiotic relationship and propose that viruses are an important part of the Hydra holobiont by controlling the species-specific microbiome. We conclude that beneficial virus-bacterial-host interactions should be considered as an integral part of animal development and evolution.

Expanding our Understanding of the Seaweed Holobiont: RNA Viruses of the Red Alga Delisea pulchra.

Marine seaweeds are holobionts comprised of the macroalgal hosts and their associated microbiota. While the composition of the bacterial component of seaweed microbiomes is increasingly studied, almost nothing is known about the presence, diversity and composition of viruses in macroalgae in situ. In this study, we characterize for the first time the viruses associated with a red macroalga, Delisea pulchra. Using transmission electron microscopy we identified diverse morphotypes of virus-like particles in D. pulchra ranging from icosahedral to bacilliform to coiled pleomorphic as well as bacteriophages. Virome sequencing revealed the presence of a diverse group of dsRNA viruses affiliated to the genus Totivirus, known to infect plant pathogenic fungi. We further identified a ssRNA virus belonging to the order Picornavirales with a close phylogenetic relationship to a pathogenic virus infecting marine diatoms. The results of this study shed light on a so far neglected part of the seaweed holobiont, and suggest that some of the identified viruses may be possible pathogens for a host that is already known to be significantly impacted by bacterial infections.

Species-specific viromes in the ancestral holobiont Hydra.

Recent evidence showing host specificity of colonizing bacteria supports the view that multicellular organisms are holobionts comprised of the macroscopic host in synergistic interdependence with a heterogeneous and host-specific microbial community. Whereas host-bacteria interactions have been extensively investigated, comparatively little is known about host-virus interactions and viral contribution to the holobiont. We sought to determine the viral communities associating with different Hydra species, whether these viral communities were altered with environmental stress, and whether these viruses affect the Hydra-associated holobiont. Here we show that each species of Hydra harbors a diverse host-associated virome. Primary viral families associated with Hydra are Myoviridae, Siphoviridae, Inoviridae, and Herpesviridae. Most Hydra-associated viruses are bacteriophages, a reflection of their involvement in the holobiont. Changes in environmental conditions alter the associated virome, increase viral diversity, and affect the metabolism of the holobiont. The specificity and dynamics of the virome point to potential viral involvement in regulating microbial associations in the Hydra holobiont. While viruses are generally regarded as pathogenic agents, our study suggests an evolutionary conserved ability of viruses to function as holobiont regulators and, therefore, constitutes an emerging paradigm shift in host-microbe interactions.

Compounds associated with algal surfaces mediate epiphytic colonization of the marine macroalga Fucus vesiculosus.

The macroalga Fucus vesiculosus carries a specific community of surface bacteria. To identify chemical compounds that possibly mediate abundance and community composition of algae-associated bacteria, we tested the effect of surface-available algal compounds on bacterial settlement and community composition under field conditions. Compounds on algal thalli were separated from the surface by extraction with organic solvents and investigated on growth inhibition and settlement of bacterial isolates. Based on in vitro data, partially purified extract fractions were then exposed to bacterial colonizers in vivo followed by bacterial enumeration and community analysis. The polar fraction of the algal surface extract revealed a significant profouling effect for Vibrionales, whereas the nonpolar fraction - containing the xanthophyll pigment fucoxanthin and other unidentified nonpolar surface compounds - revealed a significant 80% reduction of surface colonizing bacteria. The analysis of bacterial surface communities by 454 pyrosequencing demonstrated that the antifouling activity of nonpolar algal surface compounds was targeting the abundance of natural bacterial colonizers rather than the relative composition of bacterial members within the community. Moreover, the bacterial community composition on F. vesiculosus was markedly different from artificial control substrates and chemically manipulated experimental treatments, suggesting that other, nonextractable surface features and/or physical properties render algal-specific epiphytic bacterial communities.

rRNA-based profiling of bacteria in the axilla of healthy males suggests right-left asymmetry in bacterial activity.

The activity of the human armpit microbiota triggers the formation of body odor. We used differential 16S rRNA gene (rDNA)- and rRNA-based terminal-restriction fragment length polymorphism fingerprinting in combination with cloning and sequencing to identify active members of the human armpit microbiota. DNA and RNA were isolated from skin scrub samples taken from both armpits of 10 preconditioned, healthy males. The fingerprint profiles indicated pronounced similarities between the armpit microbiota in the right and the left axillae of an individual test person, but larger differences between the axilla microbiota of different individuals. Using 16S rDNA and rRNA sequence data, the majority of peaks in the armpit profiles were assigned to bacteria affiliated with well-known genera of skin bacteria. The relative abundances of all groups were similar among the rDNA and rRNA samples, suggesting that all groups of armpit bacteria were active. Surprisingly, the relative abundance of sequences affiliated with Peptoniphilus sp. was by far and with statistical significance the highest in the rRNA samples of the right armpits. Thus, bacteria affiliated with Peptoniphilus sp. might have been particularly active in the right axillae of the test persons, possibly owing to the handedness of the test persons, which might cause different environmental conditions in the right axillae.

Epibacterial community patterns on marine macroalgae are host-specific but temporally variable.

Marine macroalgae are constantly exposed to epibacterial colonizers. The epiphytic bacterial patterns and their temporal and spatial variability on host algae are poorly understood. To investigate the interaction between marine macroalgae and epiphytic bacteria, this study tested if the composition of epibacterial communities on different macroalgae was specific and persisted under varying biotic and abiotic environmental conditions over a 2-year observation time frame. Epibacterial communities on the co-occurring macroalgae Fucus vesiculosus, Gracilaria vermiculophylla and Ulva intestinalis were repeatedly sampled in summer and winter of 2007 and 2008. The epibacterial community composition was analysed by denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene libraries. Epibacterial community profiles did not only differ significantly at each sampling interval among algal species, but also showed consistent seasonal differences on each algal species at a bacterial phylum level. These compositional patterns re-occurred at the same season of two consecutive years. Within replicates of the same algal species, the composition of bacterial phyla was subject to shifts at the bacterial species level, both within the same season but at different years and between different seasons. However, 7-16% of sequences were identified as species specific to the host alga. These findings demonstrate that marine macroalgae harbour species-specific and temporally adapted epiphytic bacterial biofilms on their surfaces. Since several algal host-specific bacteria were highly similar to other bacteria known to either avoid subsequent colonization by eukaryotic larvae or to exhibit potent antibacterial activities, algal host-specific bacterial associations are expected to play an important role for marine macroalgae.