Search

Probiotics are intended to improve gastrointestinal health when consumed. However, the probiotics marketed today only colonize the densely populated gut to a limited extent. Bacteriophages comprise the majority of viruses in the human gut virome and there are strong indications that they play important roles in shaping the gut microbiome. Here, we investigate the use of fecal virome transplantation (FVT, sterile filtrated feces) as a mean to alter the gut microbiome composition to lead the way for persistent colonization of two types of probiotics: Lacticaseibacillus rhamnosus GG (LGG) representing a well-established probiotic and Akkermansia muciniphila (AKM) representing a putative next-generation probiotic. Male and female C57BL/6NTac mice were cohoused in pairs from 4 weeks of age and received the following treatment by oral gavage at week 5 and 6: AKM+FVT, LGG+FVT, probiotic sham (Pro-sham)+FVT, LGG+Saline, AKM+Saline, and control (Pro-sham+Saline). The FVT donor material originated from mice with high relative abundance of A. muciniphila. All animals were terminated at age 9 weeks. The FVT treatment did not increase the relative abundance of the administered LGG or AKM in the recipient mice. Instead FVT significantly (p < 0.05) increased the abundance of naturally occurring A.muciniphila compared to the control. This highlights the potential of propagating the existing commensal “probiotics” that have already permanently colonized the gut. Being co-housed male and female, a fraction of the female mice became pregnant. Unexpectedly, the FVT treated mice were found to have a significantly (p < 0.05) higher fertility rate independent of probiotic administration. These preliminary observations urge for follow-up studies investigating interactions between the gut microbiome and fertility.

Necrotizing enterocolitis (NEC) is a life-threatening gastrointestinal disorder afflicting preterm infants, which is currently unpreventable. Fecal microbiota transplantation (FMT) is a promising preventive therapy, but the transfer of pathogenic microbes or toxic compounds raise concern. Removal of bacteria from donor feces by micropore filtering may reduce this risk of bacterial infection, while residual bacteriophages could maintain the NEC-preventive effects. We aimed to assess preclinical efficacy and safety of fecal filtrate transplantation (FFT). Using fecal material from healthy suckling piglets, we compared rectal FMT administration (FMT, n = 16) with cognate FFT by either rectal (FFTr, n = 14) or oro-gastric administration (FFTo, n = 13) and saline (CON, n = 16) in preterm, cesarean-delivered piglets as models for preterm infants. We assessed gut pathology and analyzed mucosal and luminal bacterial and viral composition using 16S rRNA gene amplicon and meta-virome sequencing. Finally, we used isolated ileal mucosa, coupled with RNA-Seq, to gauge the host response to the different treatments. Oro-gastric FFT completely prevented NEC, which was confirmed by microscopy, whereas FMT did not perform better than control. Oro-gastric FFT increased viral diversity and reduced Proteobacteria relative abundance in the ileal mucosa relative to control. An induction of mucosal immunity was observed in response to FMT but not FFT. As preterm infants are extremely vulnerable to infections, rational NEC-preventive strategies need incontestable safety profiles. We show in a clinically relevant animal model that FFT, as opposed to FMT, efficiently prevents NEC without any recognizable side effects.

Bacterial vaginosis (BV) is characterized by a reduction in Lactobacillus (L.) spp. abundance and increased abundance of facultative anaerobes, such as Gardnerella spp. BV aetiology is not fully understood; however, bacteriophages could play a pivotal role in the perturbation of the vaginal bacterial community. We investigated the vaginal viral community, including bacteriophages and the association to the bacterial community and BV-status. Vaginal samples from 48 patients undergoing IVF treatment for non-female factor infertility were subjected to metagenomic sequencing of purified virus-like particles. The vaginal viral community was characterized and correlated with the BV-status by Nugent score, bacterial community, structure, and the presence of key vaginal bacterial species. The majority of identified vaginal viruses belonged to the class of double-stranded DNA bacteriophages, with eukaryotic viruses constituting 4% of the total reads. Clear links between the viral community composition and BV (q = 0.006, R = 0.26) as well as the presence of L. crispatus (q = 0.001, R = 0.43), L. iners, Gardnerella spp., and Atopobium vaginae were found (q < 0.002, R > 0.15). The eukaryotic viral community also correlated with BV-status (q = 0.018, R = 0.20). In conclusion, the vaginal virome was clearly linked with bacterial community structure and BV-status.

To investigate the notion that starter cultures can be a reservoir of bacteriophages (phages) in the dairy environment, strains of three DL-starters (undefined mesophilic mixed-strain starters containing Lactococcus lactis subsp. lactis biovar. diacetylactis and Leuconostoc species) were selected and induced by mitomycin C, and the whole starters were induced spontaneously as well as by mitomycin C. Frequency of induction of 17%, 26% and 12% was estimated among the isolates of the three starters, with majority of the induced phages mostly showing morphological similarity to known P335 phages, and with a fraction of them showing atypical features. Sequences of P335 quasi-species phages were found to be the most frequent entities in almost all metaviromes derived from the induced lysates. However, sequences of Sk1virus phages (previously 936 phages) were emerged as the predominant entities following spontaneous induction of one of the starters, suggesting a phage-carrier state. Sequences of other phages such as 949, 1706, C2virus (previously c2 phages) and Leuconostoc species could also be observed but with a lower relative frequency. Taken together, the majority of the P335 quasi-species phages could represent the induced viral community of the starters and the remaining phage groups mainly represent the background ambient viral community.

Changes of microbial communities have been linked to frailty in elderly, yet the presence of fungal communities and their associations are little understood. This study attemps to identify biologically meaningful gut microbial fungal associations during aging. Faecal samples of 100 Danes of 65 years or older were collected, and sequenced by high-throughput tag-encoded sequencing of ITS2 gene fragments. The sequences were analysed using QIIME and CoNet to characterise fungal communities, and generate association networks, respectively. HbA1c, identified as the principal node, was grouped into 3 clusters based on glycated glucose levels. The clusters correspond to the phyla Ascomycota, Basidiomycota and Zygomycota, with the genera Penicillium, Candida, and Aspergillus being particularly abundant within each cluster. Interestingly, Bray-Curtis dissimilarity matrices showed significant (P<0.05) variation between clusters. These findings suggest that the presences of specific gut mycobiome members are associated with glycemic behaviours among the healthy individuals of the elderly Danish population.

The human gastrointestinal tract (GIT) is inhabited by a vast amount of microorganisms from different domains of life collectively denominated the gut microbiome (GM). Among its numerous functions, GM plays a crucial role in developing the immune system in early-life and contributes to maintain a balanced metabolism later in life. During the last decade, studies have revealed that imbalances in GM composition, typically known as dysbiosis, are able to trigger metabolic (and immunological) abnormalities in the host. This has been demonstrated to have implications on the development of metabolic disorders, such as obesity, elevated blood pressure, high serum triglycerides, low high-density lipoprotein (HDL) levels and insulin resistance, which constitute a cluster of risk factors for development of the so-called metabolic syndrome (MetS). In turn, these disorders may also influence muscle composition, physiological decline and frailty in older individuals (e.g. +65 years). Identifying lifestyle factors and their interactions with GM and host would be of great societal value in relation to preventing frailty and improving the life quality of the individuals, as well as for economical reasons. Our work gathers an overview on current methodologies for GM screening, with particular emphasis on viral communities, it presents an integrative approach to elucidate the interplay of dietary aspects and GM composition in relation to physiological decline, and finally, it provides insights of the putative role of viral communities in modulating GM and metabolic biomarkers in older subjects. In the first manuscript, we assessed and optimized two methods for phageextraction based on tangential-flow filtration (TFF) and polyethylene glycol precipitation (PEG). Through our improved procedures, we reported significantly higher yields of extracellular viral-like particles (eVLPs) and their DNA

Despite their huge potential for characterizing the biodiversity of phages, metagenomic studies are currently not available for dairy bacteriophages, partly due to the lack of a standard procedure for phage extraction. We optimized an extraction method that allows to remove the bulk protein from whey and milk samples with losses of less than 50% of spiked phages. The protocol was applied to extract phages from whey in order to test the notion that members of Lactococcus (Lc.) lactis 936 (now Sk1virus ), P335, c2 (now C2virus ) and Leuconostoc phage groups are the most frequently encountered in the dairy environment. The relative abundance and diversity of phages in eight and four whey mixtures from dairies using undefined mesophilic mixed-strain DL-starter cultures and defined cultures, respectively, was assessed. Results obtained from transmission electron microscopy and high-throughput sequence analyses revealed the dominance of Lc. lactis 936 phages (order Caudovirales, family Siphoviridae ) in dairies using undefined DL-starter cultures and Lc. lactis c2 phages (order Caudovirales, family Siphoviridae ) in dairies using defined cultures. The 936 and Leuconostoc phages demonstrated limited diversity. Possible co-induction of temperate P335 prophages and satellite phages in one of the whey mixtures was also observed.

Microbiomes of the neritic copepod species Acartia tonsa and Temora longicornis collected in coastal Danish waters were investigated by use of 16S rRNA gene-Amplicon highthroughput sequencing. In contrast to current assumptions and findings, microbiomes of the 2 copepod species from the same environment were significantly different in bacterial species composition, particularly in terms of relative abundance of bacteria associated with each copepod species. The microbiomes of both species, likewise, differed from that of A. tonsa reared in laboratory culture, but similarities were also observed between wild and laboratory-reared A. tonsa. Alpha proteobacteria dominated A. tonsa microbiomes, whereas Gammaproteobacteria were most abundant in T. longicornis microbiomes. The overall diversity of bacteria associated with the 2 copepod species was to some extent in harmony with previous findings, but several of the most abundant operational taxonomic units correspond to bacteria that have not previously been found in association with marine copepods. The results thus add to the knowledge of the diversity of microbiomes of marine zooplankton organisms and document the fact that different planktonic copepod species from the exact same environment may host significantly different microbiomes. Hence, different copepod species are either not colonized by the same bacteria assemblages or support colonization/growth of different bacteria populations, leading to distinct copepod microbiomes.

Aging is associated with changes of the prokaryotic component of the gut microbiota - becoming less diverse, and increasing proinflammatory profile compared to younger adults. These changes have been linked with frailty in several studies. However, our knowledge of the influence of the gut mycobiome on health and disease in elderly remain sparsely investigated. Consequently, the aim of this study was to characterise the feacal mycobiota in relation to host health parameters.Feacal samples from 99 healthy individuals ranging from 65 to 81 years old were collected, and fungal composition was determined using ITS2 amplicon sequencing. The obtained sequences were analysed non-parametrically using the QIIME pipeline to assess fungal taxa composition and diversity based on alpha and beta diversity, respectively. ANOSIM and adonis analyses were performed to assess the significance level between categories associated with the clinical features among individuals.The elderly gut is home to three main phyla Ascomycota, Basidiomycota and Zygomycota, with genera Penicillium, Candida, and Aspergillus being particularly common. Based on HbA1c-levels, the individuals could be clustered into 3 groups, High, Medium and Healthy. Clusters according to genus abundance co-segregated with glycated glucose levels (HbA1c), carbohydrate intake, and insulin secretion based on C-peptide levels. Interestingly, the dissimilarity matrices of Bray-Curtis showed significant (P<0.05) variation between clusters with glycated glucose level.Collectively, these findings suggest that the presences of specific gut mycobiome member is associated with glycemic behaviours among the healthy individuals of the elderly Danes population.

Background Aging is associated with decreased muscle mass and functional capacity, which in turn decrease quality of life. The number of citizens over the age of 65 years in the Western world will increase by 50 % over the next four decades, and this demographic shift brings forth new challenges at both societal and individual levels. Only a few longitudinal studies have been reported, but whey protein supplementation seems to improve muscle mass and function, and its combination with heavy strength training appears even more effective. However, heavy resistance training may reduce adherence to training, thereby attenuating the overall benefits of training. We hypothesize that light load resistance training is more efficient when both adherence and physical improvement are considered longitudinally. We launched the interdisciplinary project on Counteracting Age-related Loss of Skeletal Muscle Mass (CALM) to investigate the impact of lifestyle changes on physical and functional outcomes as well as everyday practices and habits in a qualitative context. Methods We will randomize 205 participants older than 65 years to be given 1 year of two daily nutrient supplements with 10 g of sucrose and 20 g of either collagen protein, carbohydrates, or whey. Further, two groups will perform either heavy progressive resistance training or light load training on top of the whey supplement. Discussion The primary outcome of the CALM Intervention Study is the change in thigh cross-sectional area. Moreover, we will evaluate changes in physical performance, muscle fiber type and acute anabolic response to whey protein ingestion, sensory adaptation, gut microbiome, and a range of other measures, combined with questionnaires on life quality and qualitative interviews with selected subjects. The CALM Intervention Study will generate scientific evidence and recommendations to counteract age-related loss of skeletal muscle mass in elderly individuals.

Copepods are to an increasing extent cultivated as feed for mariculture fish larvae with variable production success. In the temperate climate zone, this production faces seasonal limitation due to changing abiotic factors, in particular temperature and light. Furthermore, the production of copepods may be influenced by biotic factors of the culture systems, such as competing microorganisms, harmful algae, or other eukaryotes and prokaryotes that may be non-beneficial for the copepods. In this study, the composition of bacteria associated with copepods was investigated in an extensive outdoor copepod production system. Light microscopy and scanning electron microscopy revealed that bacteria were primarily found attached to the exoskeleton of copepods although a few bacteria were also found in the gut as well as internally in skeletal muscle tissue. Through 16S rRNA gene-targeted denaturing gradient gel electrophoresis (DGGE) analysis, a clear difference was found between the microbiomes of the two copepod species, Acartia tonsa and Centropages hamatus, present in the system. This pattern was corroborated through 454/FLX-based 16S rRNA gene amplicon sequencing of copepod microbiomes, which furthermore showed that the abiotic parameters pH and oxygen concentration in rearing tank water were the key factors influencing composition of copepod microbiomes.