Significance of the Gut Microbiome for Viral Diarrheal and Extra-Intestinal Diseases

The composition of the mammalian gut microbiome is very important for the health and disease of the host. Significant correlations of particular gut microbiota with host immune responsiveness and various infectious and noninfectious host conditions, such as chronic enteric infections, type 2 diabetes, obesity, asthma, and neurological diseases, have been uncovered. Recently, research has moved on to exploring the causalities of such relationships. The metabolites of gut microbiota and those of the host are considered in a ‘holobiontic’ way. It turns out that the host’s diet is a major determinant of the composition of the gut microbiome and its metabolites. Animal models of bacterial and viral intestinal infections have been developed to explore the interrelationships of diet, gut microbiome, and health/disease phenotypes of the host. Dietary fibers can act as prebiotics, and certain bacterial species support the host’s wellbeing as probiotics. In cases of Clostridioides difficile-associated antibiotic-resistant chronic diarrhea, transplantation of fecal microbiomes has sometimes cured the disease. Future research will concentrate on the definition of microbial/host/diet interrelationships which will inform rationales for improving host conditions, in particular in relation to optimization of immune responses to childhood vaccines.


Introduction
The human gut microbiota, comprising bacteria, viruses, fungi, protozoa, and parasites and comprehensively termed the gut microbiome, have received increased attention for about a decade, when it was recognized that their commensal or symbiotic relationship is of great importance for human health, including immune responses correlated with protection from infection or disease [1][2][3][4]. Most of the microbiome data relate to the bacteria (bacteriome) and viruses (virome) populating the gut. Observational studies initially reported on cotemporal correlations of the composition of the gut microbiome with immune responses or disease outcome [5,6]. More recent studies aimed at identifying causal relationships between metabolic products of the gut microbiome and the host in health and disease [7][8][9]. This review emphasizes the importance of the transition from observational correlation studies to studies exploring causal microbiome-host relationships, which will provide data for rational developments of microbiota as probiotic agents.

The Intestinal Microbiome
The main bacterial phyla present in the gut are Proteobacteria, Bacteriodetes, Firmicutes, and Actinobacteria, with their total number estimated to be 10 14 particles, and populating mainly the colon [10,11]. The gut microbiota in infants are originally similar to those of the mother but will develop by colonization with Bifidobacterium, Bacteroides, and Clostridium spp. [12], and the composition of the gut microbiome will highly depend on nutritional/feeding and environmental conditions [4,13]. Viruses found in the gut are mainly members of the Picornaviridae, Reoviridae, Caliciviridae, and Astroviridae families, and of various families of bacteriophages; in addition, members of the Adenoviridae, From observational studies, it has been recognized that the immune responses of children to oral or parenteral vaccines in low-income countries are often weak and that this finding correlated with the particular composition of the gut bacteriome of these children [31]. The presence of Clostridia and Proteobacteria correlated with a favorable immune response to rotavirus vaccination in Pakistan [32]. In Ghana, enrichment of Bifidobacteria was associated with a favorable immune response of children and that of Enterobacteria and Pseudomonas with low immune responses and lower protection [33]. The composition of the microbiome of high responders was similar to that of high responders in high-income countries [32,33]. No such differences were found in children receiving rotavirus vaccination in Nicaragua [34]. However, most evidence indicates that the composition of the intestinal microbiome is important for the improvement of vaccine efficacies [24,35]. The reasons for decreases in immune responses are complex. Besides the composition of the gut microbiome, malnutrition (including zinc deficiency and vitamin A and D avitaminoses), intestinal and extraintestinal coinfections, immunological immaturity (often linked with premature birth), the presence of maternal antibodies (transmitted via placenta), and host genetic factors play a role [3,36,37].

In Animals
The influence of the gut microbiome on enteric infections has been extensively studied in animal models. Thus, gnotobiotic (gn) piglets transplanted with 'healthy' human gut microbiota from children (HHGM: Proteobacteria, Bacteriodetes) or with microbiota from children with weak ('unhealthy') immune responses (UHGM: Proteobacteria and Firmicutes) differed in their reaction to challenge with human rotavirus: the HHGM-transplanted animals expanded Bacteriodetes and had less severe diarrhea and virus shedding than UHGM-transplanted animals, which maintained the high prevalence of Firmicutes spp. [38]. Similarly, neonatally GF piglets transplanted with a human infant's fecal microbiome (HIFM) upon challenge had less severe rotavirus disease than nontransplanted piglets; a protein-deficient diet increased the severity of RV disease also in the HIFM-transplanted piglets [39].
In both human and animal vaccine studies, it has been shown that the composition of the gut microbiome is highly important for the efficacy of vaccines, e.g., against RV, poliovirus, and bacteria, such as Salmonella and Shigella [6,40].

Intestinal Microbiome-Host Interaction via Metabolites
In animal studies, it has become apparent that differences in the metabolism of bacteria may be important for the host's health. Bacterial metabolites produced in the gut may enter the host via hematogenic spread and either be toxic or support the health of the gut or extraintestinal tissues. Metabolites of both microbes and host form a complex system, and humans have been termed as 'holobionts' in this concept [9]. Experiments aiming at discovering causal microbiome-host relationships were initiated [7]. Numerous microbial metabolites were shown to affect the host's metabolic pathways and to be positively or negatively associated with metabolic diseases such as type 2 diabetes (T2D) or obesity [7]. For the microbiome-host relationship, the supply of polymeric compounds for bacterial fermentation in particular diets plays a very important role [8]. Some products of bacterial fermentation and selected bacterial species producing them are listed in Table 1. Of those metabolites:

1.
Short-chain fatty acids (SCFAs) have anti-inflammatory activity [41] and can act as adjuvants to vaccines [42]; butyrate-producing bacteria were found to be beneficial as probiotics (see below) in children with idiopathic nephrotic syndrome by boosting the synthesis of Treg cells [43]; 2.
Products of histidine fermentation may cause immune pathologies and be involved in asthma pathogenesis [49,50]; 5.
Imidazole propionate production is correlated with an increased risk for the development of T2D [51,52]; 6.
Dopamine is generated by bacterial decarboxylases from levodopa, used for the treatment of Parkinson's disease [53,54]; 7.
Host bile acids are deconjugated and transformed into secondary bile acids in the colon, where they can inhibit the growth of Clostridioides difficile [57], but are also associated with an increased risk of obesity development [58]; 10. Trimethylamine-N-oxide, derived from bacterial metabolization of choline and carnitine, has been found to be associated with a risk to develop atherosclerosis and T2D [59][60][61]; 11. Sphingolipids derived from Bacteroides spp. metabolism are important for intestinal bacterial homeostasis [62,63]. The interplay between diet, gut microbiota fermentation, and host cellular pathways leads to a complex microbe-host-produced spectrum of metabolites, strongly suggesting that the gut microbiome affects the host's health in a much more general way than just by its influence on immune responses.

Intestinal Microbiome and Diet
In studying causal relationships between the gut microbiome composition and the mammalian host's health phenotype, the transplantation of human gut microbiome to experimental animals has encountered the problem that not all human bacteria may survive in the new host, also due to the fact that animal and human diets differ substantially. This has been clearly demonstrated in a study by Rodriguez et al. [67], who showed that the basal diet of mice determined the long term composition of their gut microbiome and the mouse phenotypes to a greater extent than the transfer of largely different fecal microbiomes obtained from lean or obese human donors.
In order to overcome some of these problems, a mouse model for the study of dietmicrobe-host interactions has been developed using the following procedure [68]:

1.
A human simplified intestinal microbiota (SIM) consisting of 10 human bacterial strains able to metabolize dietary fibers was constructed; 2.
SIM bacteria were transferred to GF mice; 3.
Mice were kept on three different diets: chow (fiber-rich), high fat-high sucrose (low in fiber), and zero fat-high sucrose (low in fiber).
The system was used to study how the different diets may affect the abundance and the transcriptome of SIM bacteria, how SIM-diet interactions may affect the circulation of metabolites, and how this may affect the metabolism of the host. Preliminary results showed that: 1.
The diet affected the SIM bacteria colonization and their fermentation capacity; 2.
Diet-SIM bacteria interaction affected the systemic entry of SIM metabolites into the plasma of the host; 3.
The host metabolism in turn depended on the diet taken.
A microbiota-directed complementary food prototype was developed for 12-18 m old malnourished children and been found to be beneficial for weight and height gain, increase in plasma protein levels, and population by Faecalibacterium and Bifidobacterium spp. [69].

Intestinal Microbiome and Infectious and Non-Infectious Diseases
Dysbiotic microbiomes can lead to intestinal infectious diseases such as inflammatory bowel disease, necrotizing enterocolitis, irritable bowel syndrome, chronic Clostridioides difficile diarrhea, and extraintestinal infectious diseases [70]. Microbiota research should focus on discovering causal links between human microbiota and infectious and immune-mediated diseases [71].
A combination of dietary conditions and altered gut microbiomes was found to be associated with T2D [52,67,72], obesity [73], nonalcoholic fatty liver disease [74,75], idiopathic nephrotic syndrome [43], and cardiovascular diseases such as hypertension [76] or atherosclerotic disease [60,61,77]. In detail, the pathogenic mechanisms are complex and often systemic. The metabolic potential of gut microbiota (see above) may generate bioactive compounds, which can interact with the host in various ways [61].

Diet, Prebiotics, and Intestinal Microbes as Probiotics
Prebiotics are components of food which support the growth of gut microorganisms beneficial for human health. They mainly consist of fibers, which nondigestible in the mammalian small intestine but suitable as substrates for bacteria in the colon, mainly Bifidobacteria and Lactobacillus. A major metabolic product of bacterial fermentation of starches is SCFAs, which have antibacterial activity [64,65]. Dietary polyphenols have been shown to have anti-inflammatory and possibly prebiotic activities [56]. Gnotobiotic mice colonized by a consortium of human gut-derived bacteria were fed different foodderived fibers; by administering retrievable artificial food particles, it was possible to identify bacterial species specialized in the degradation of particular types of fiber [78]. Analysis of the microbiota of a healthy Bangladeshi birth cohort enabled the identification of several covarying bacteria-potentially leading the way toward gut microbiota repair in undernourished children [79].
Symbiotics are defined as a mixture of beneficial bacteria (probiotics) and fibers (prebiotics) on which the bacteria feed. They can be used as food supplements, typically consisting of lactic acid bacteria (Lactobacillus paracasei, L. plantarum) and plant fibers (pectin from citrus fruits, inulin from chicory root, starch from corn) and are applied as a remedy for microbe-associated acute infantile and noninfectious chronic diarrheas [92].

Intestinal Microbes as Therapeutics
Fecal microbiota transplantation (FMT) is an established procedure to treat chronic, AB-resistant, Clostridioides difficile-associated chronic diarrhea [93,94]. However, the overall rate of clinical cure is variable, and major adverse clinical events are not rare [7,95]. Careful risk assessment is indicated [96].

Outlook and Future Research
Knowledge of the gut microbiome, including its establishment, evolution, changes, and association with intestinal and extraintestinal diseases has enormously increased during the past 10 years. In particular, the relationship between particular gut microbiome compositions and immune responsiveness to invading microbes or vaccines has been analyzed. However, increasingly, observational studies aiming at cotemporal correlations of microbiome composition and clinical symptoms have given way to investigations in which causal relationships between gut microbiome composition, diet, complex metabolic end products (of both the microbiome and the mammalian host), and clinical phenotypes are being explored. Such data will form a rational basis for the use of gut microbiomes as therapeutic or probiotic agents.
A list of topics that remain open for future research has been collated in Table 2. The molecular biology of the interaction of gut microbiome and host, the dependence of microbiota on diet, and the influence of the joint microbiome-host metabolome on health and disease require more detailed understanding and study. Factors determining eubiosis/dysbiosis in the microbiome-host relationship have to be identified in relation to host clinical phenotypes, particularly in children from low-income countries. Mechanisms determining the influence of probiotic bacterial metabolites on the host's immune responses have to be defined. The use of probiotic bacteria for the improvement of extended programs of immunization (EPI) in low-and middle-income countries has to be explored and optimized. Table 2. Topics of future research on gut microbiota.

Molecular Biology
Interrelationship of host and gut microbiota metabolism and influence of nutrition Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.

Conflicts of Interest:
The author declares no conflict of interest.