The aims of the present study were to determine (1) whether bacteriophages could induce alterations of the gut flora, leading to gut leakiness and an impaired intestinal barrier; and (2) whether bacteriophage treatment could increase gut permeability in mammals, sufficient to facilitate endotoxemia and inflammatory responses.

Our knowledge of the particular role of intestinal permeability has greatly increased over the last years24, 25. Indeed, with the impaired gut permeability, bacterial antigens cross into the lamina propria, leading to endotoxemia and dysregulation of inflammatory responses, and trigger chronic inflammation, which in turn is an important factor for the development of various polyaetiological diseases20, 21. There is increased evidence showing that the concomitant gut barrier dysfunction may play a critical role in diseases such as Crohn disease, IBD, chronic fatigue syndrome, diabetes, autism, cancer, neurodegenerative and cardiovascular diseases3,4,5,6,7. At the same time, alterations in the gut microbiota, which is an important regulator of intestinal homeostasis, have been proposed to play a primary role in the impaired gut permeability and deleterious immune activation2. Therefore, understanding all the factors that modulate microbiota is essential for the prevention and therapy of a variety of poorly treatable diseases.

Bacteriophages are known to play an important role in microbiota homeostasis and therefore are involved in human health26. De Paepe et al. described different models including both direct and indirect effects of phages on humans. Indirect models are predominantly realized by the role of phages in dysbiosis, alterations of bacterial properties by transduction, and phage-mediated changes in recognition patterns of bacteria, resulting in an altered immune response. Direct models describe the direct modulation of immunity resulting from phage phagocytosis27.

Our previous results have indicated, for the first time, that bacteriophages might be a previously unrecognised cause of diseases in mammals, including humans. To our knowledge, our previous study was the first to indicate that bacteriophage treatment could lead to intestinal hyperpermeability in rodents14. We have shown that the challenge of animals with bacteriophages resulted in increased intestinal permeability and elevation of circulating immune complexes. This observation led us to define such alterations as ‘microbiota diseases’, which reflects the nature of the primary impairment caused by bacteriophages.

In the present study, we used the same rat model and treatment regimen to study, in more detail, the changes in the gut microbiota and endotoxemia following bacteriophage treatment. To understand the nature of impaired gut permeability in response to bacteriophage exposure, faecal material was collected from the same animals before and after the phage challenge. We used a bacteriophage cocktail comprised of commercially available and characterised phages active against the Enterobacteriaceae, Staphylococcaceae, Streptococcaceae, and Pseudomonadaceae families. Bacteriophages are known to selectively interact with bacteria and not affect eukaryotic cells. Thus, the impaired gut permeability can only be a consequence of altered microbiota12, 28.

We found an increased lactulose/mannitol ratio compared with that at baseline, which was determined prior to administration of the bacteriophages, indicating increased intestinal permeability following oral administration of bacteriophages. Our results also showed significantly elevated levels of blood serum endotoxin, TNF-α, IL-1β, and IL-6 compared to those in the pre-treatment period.

Consistent with other studies, which indicated that the alteration of gut microbiota by antibiotics might lead to impaired gut permeability, we showed for the first time that bacteriophages could induce inflammation, most likely through an increase in the circulating endotoxin level, which is likely to be a result of an altered intestinal microbiota and increased intestinal permeability29. There are different ways by which a disrupted gut barrier can lead to immune modulation; the most well described is stimulation of inflammatory responses by the intestinal-derived endotoxin (increased serum LPS levels), which subsequently results in elevated levels of inflammatory mediators18, 30.

In this study, we found that the bacteriophage challenge affected the microbial alpha diversity. Overall, we detected an increase in the richness and diversity of faecal microbiota, indicating that the total number of bacterial species increased after phage treatment compared to the baseline pre-treatment data. The increased richness of intestinal microbiota is considered one of the signatures of a leaky gut and a feature of intestinal inflammation. Similar patterns have been described in other diseases associated with increased intestinal permeability31, 32. Our study identified bacteria whose abundance changed following the bacteriophage challenge, and this alteration resulted in increased intestinal permeability, since phages selectively target bacteria with no effect on mammalian cells33, 34.

At the genus level, samples collected after the treatment with bacteriophages showed increases in Oscillospira and Butyrivibrio and decreases in Lactobacillus and Faecalibacterium; however, only Lactobacillus showed a two-fold change. The decreases in Lactobacillus and Faecalibacterium represent an important pattern, regarded as a signature of impaired gut permeability and inflammation35, 36, 37. Both bacterial genera are known to be beneficial for mammals, and studies have shown that their depletion is associated with barrier abnormalities. In a number of studies, both bacterial genera, which are considered effective anti-inflammatory microorganisms, were shown to restore the function of the intestinal barrier. Thus, Lactobacillus rhamnosus CNCM I-3690 and the commensal bacterium Faecalibacterium prausnitzii A2-165 exhibited similar protective effects against induced barrier hyperpermeability in mice.

It is interesting to note that the increase in relative abundance of the butyrate-producing bacteria Butyrivibrio, known to lead to decreased expression of proinflammatory cytokines and suppression of proinflammatory responses38, 39. Butyrivibrio are known to reduce bacterial translocation by potentiation of mucin synthesis, are beneficial for tight junctions, and are suggested to suppress intestinal hyperpermeability40.

Of note, the bacteriophages used in this study had no direct impact on the genera Lactobacillus, Faecalibacterium, and some others whose abundance was significantly altered. This is consistent with previous studies, which have shown that microbiota is characterized by temporal stability and dynamic equilibrium and its alterations result in complex and poorly predicted responses and consequences41.

It is likely that the decrease in the abundance of certain genera, which were directly affected by the phages, triggered a cascade of microbiota alterations, eventually leading to the depletion of Lactobacillus and Faecalibacterium species. This may be the result of alteration of the abundance of species having symbiotic associations with Lactobacillus and Faecalibacterium or of outgrowth of species having inhibitory interactions with these two species42.

An example of possible alterations of symbiotic properties is the decreased relative abundance of Streptococcus spp that are known to have a mutualistic relationship with certain Lactobacillus spp as a result of the direct impact of phages used in this study43.

In contrast, one of the possible inhibitory pathways may involve increased abundance of Helicobacter spp., whose antagonistic effects towards Lactobacillus were previously described44, 45. In turn, the abundance of Helicobacter is likely to be increased by the decrease of Enterobacteriacea, which is directly affected by the bacteriophage cocktail46.

On the other hand, participation of these species in the gut balance is well known, while relative contributions of other bacteria, whose abundance also changed in this study and which could regulate the intestinal barrier function, may be underestimated. Therefore, at this point the exact pathway of microbiota alteration that leads to gut leakiness remains unknown. However, this was not the goal of our study since bacteriophages are known to specifically target specific bacterial species, and therefore, other types of phages may lead to different microbiota alterations and consequences for the macroorganism.

The experimental model used allows to minimize possible direct effect of used phages on mammalian intestinal epithelium as there are numerous of available receptors of specific bacteriophage receptors on the surface of the bacteria of rats’ microbiota.

Collectively, our results warrant further research on microbiota diseases and previously underestimated consequences related to bacteriophage exposure in mammals, including humans. We suggest that the global distribution of bacteriophages, their high prevalence in the outer environment and microbiota, and their potential to alter gut permeability, which was revealed in this study, should drive future research regarding phage implications in a variety of emerging diseases. Although it is not possible to determine, based on this study, if the observed alterations occur in the natural environment, one may assume that under certain conditions, bacteriophage infections could be contagious and spread among mammalian hosts, triggering polyaetiological conditions. Follow-up studies on the role of bacteriophages should be considered for better understanding of the implication of phages in mammalian diseases and the spread of poorly treated diseases, associated with alterations of microbiota, in humans.