Background
There is growing interest in understanding the effects of malnourishment in infancy and subsequent implications later in life [
1‐
3]. Human breast milk is a nutritious complete food and it is considered as a ‘gold’ standard for infant nutrition [
4,
5]. In conditions where breast-feeding is not possible or breast milk is not available in adequate quantities, infant formula provides an alternative safe and nutritious diet for infants [
3]. In developing countries, deprivation of nutritious diet (infant formula or breast milk) due to various reasons (sanitation, infection, poverty etc) frequently leads to malnourishment of infants [
6]. Malnutrition has devastating health consequences and increases the probability of contracting life-threatening diseases such as diarrhea, measles, pneumonia, malaria, and human immunodeficiency syndrome [
7]. Malnutrition and enteric diseases form a vicious cycle because enteric diseases are more likely to occur in a malnourished host, and enteric pathogens aggravate malnutrition symptoms. This vicious cycle is difficult to overcome without proper intervention [
8,
9]. Gastrointestinal infections affect the nutritional status due to mal-absorption of dietary intake, electrolyte imbalance, and secretory diarrhea, which lead to severe dehydration and malnourishment [
8]. On the other hand, malnutrition results in intestinal dysbiosis, sub-optimal immune function, and increased gut permeability leading to a higher probability of translocation of opportunistic pathogenic bacteria or pathobionts and secondary infections [
8,
9]. On either side of the vicious cycle ‘infection or malnutrition’, the gut microbiota acts as a bridge communicating responses and modulating the host metabolism [
10]. The intestinal microbiota plays an important role in orchestrating host health. It supports host defense and homeostasis in recovery from enteric infections [
11]. Abiotic or biotic stresses reduce the functionality of the microbiome and lower the production of metabolites usable by host [
10]. It is now evident that the composition and activities of the gut microbiota drive various local and systemic effects [
12]. Factors like xenobiotics (eg. probiotics, prebiotics or antibiotics) and enteric pathogens (eg human rotavirus, HRV) are also known to perturb the gut microbiota [
12‐
14]. With the advent of next generation sequencing technology and the availability of bioinformatic tools, numerous studies have explored microbial ecology and the relevant microbiota functions in the host [
12,
15‐
18]. For example, HRV infected infants displayed a reduction in the fecal microbiota diversity compared to healthy infants [
19]. Thus, the role of the gut microbiota is increasingly recognized in health and disease.
HRV gastroenteritis is a vaccine preventable disease in infants that accounts for approximately 215,000 deaths annually worldwide [
20]. The majority of these deaths occur in developing countries where malnourishment is common [
20]. Although vaccines are available, their efficacy is low in developing countries [
21,
22]. The poor efficacy of HRV vaccines in developing countries is attributed to numerous reasons including malnutrition and the dysbiotic gut microbiota [
21,
23,
24]. Malnutrition perturbs the gut microbiota and thereby induces negative effects on the host immune system. Therefore, malnutrition is likely to contribute to HRV vaccine failure in developing countries [
24,
25]. Identifying the specific microbial structure and composition in malnutrition and/or HRV infection has both diagnostic and therapeutic value, but not yet been fully investigated.
Due to various confounding factors and ethical concerns, addressing these questions in human infants is not possible. Human microbiota transplanted (microbiota humanized) animal models are used whereby selective microbial communities can be modeled under controlled conditions; however, not all microbiota humanized animal models recapitulate most of the donor microbiota (mouse microbiota humanized model) [
26,
27]. Numerous publications have suggested pigs as a biologically relevant and non-primate model for transplanting human gut microbiota compared to rodent models [
28‐
30]. Pigs are more advantageous non-primate models to study human conditions than rodents, because pigs are more closely related to humans in terms of anatomy, genetics, physiology and immunology and they are omnivores and outbred like humans [
27,
31]. Transplantation of the human microbiota into germfree (GF) piglets resulted in comparable microbial community structure to the original specimen [
26,
27,
32]. In contrast, humanizing GF mice with human microbiota did not recapitulate most of the microbial profiles seen in the original human donor stool [
18,
33]. Therefore, GF piglets transplanted with human intestinal microbiota are increasingly recognized as a clinically relevant model to investigate the effects of diets and enteric pathogens on the intestinal microbiota [
30,
34]. Importantly, GF pigs infected with HRV exhibit clinical signs and intestinal lesions similar to those seen in human infants, unlike the lack of HRV lesions and clinical disease in adult mouse models [
27,
35]. We hypothesized that the transplantation of human infant fecal microbiota (HIFM) into GF pigs would result in a similar assembly and composition of microbiota in the gut and furthermore, malnutrition would alter the gut microbiota leading to sub-optimal functioning of the immune system, and exacerbating HRV disease severity.
In the present study, we transplanted GF pigs with HIFM and evaluated the impact of diet on gut microbiota composition and HRV disease susceptibility. Our results indicated that HIFM pigs on a malnourished diet displayed clinical symptoms mimicking the symptoms in malnourished infants and characterized by alteration of the gut microbiota and increased susceptibility to HRV disease.
Discussion
Rotavirus accounts for up to 40% of infant diarrheal deaths [
53] and combined with an imbalanced nutrition, rotavirus is one of primary causes of mortality and morbidity worldwide [
54]. An infectious dose as low as 10 virulent HRV particles is sufficient to infect and cause diarrhea in a susceptible individual. Once a child is infected, he can spread the infection up to 50% of the children in close contact, increasing HRV incidence [
55‐
58]. Hence, the amount of HRV shedding and the duration of shedding in infected individuals are of paramount importance in HRV diarrheal outbreaks. Nutritional status and gut-microbiota play significant roles in maintaining gut barrier function [
9,
59,
60]. Perturbations of these two parameters have additive effects on the persistence of malnutrition and enteric infections [
9,
61]. The triad of ‘diet-gut microbiota-host response’ is important in an individual’s overall development but more importantly in infants due to the recent concept of ‘the first 1000 days of life’ [
62].
Although, not many studies have investigated the impact of diet, HRV infection, and gut microbiota in humans, only a few studies (including ours) have tried to mimic the human infant microbiome in animal models, using neonatal GF piglet transplanted with HIFM to study these parameters [
50]. Our pilot study showed that at PTD7 more than 99% of the bacterial diversity present in the original HIFM fecal samples from a two-month-old baby was represented all along the pig intestines and in feces. Different proportions of bacteria were detected in the tissues studied, suggesting that some bacteria grow better in pigs depending on the intestinal location. For example,
Bifidobacterium was present in higher abundance in the original HIFM fecal samples and 10 times less in the HIFM pig intestinal and fecal samples. The original HIFM sample was obtained from a breast fed baby, while HIFM pigs were formula fed.
Bifidobacterium are frequently transferred from mother-to-infant, and it has been shown that breast-feeding increases the diversity and abundance of
Bifidobacteria [
63‐
65]. As expected, some bacteria not detected in the original HIFM fecal sample were detected in the HIFM transplanted pigs. However, these bacteria were lower than 0.7% in each pig tissue relative to the whole microbiota. It is likely that these unique bacteria were at very low concentration in the original HIFM fecal sample and were not detectable after sample processing for metagenomics studies. The diet may have contributed to the enrichment of these bacteria in pig gut. This was supported by the presence of unique bacteria mostly in the upper part of the intestine and less in the lower part. Despite these variations in the microbial population, our results suggested that 7 days are sufficient to have a representative colonization of the pig intestines by the original HIFM.
HRV infected malnourished piglets had significant reduction in body weight gain and an enhanced diarrhea [
50]. A recent study also showed that malnutrition was significantly associated with more severe HRV induced diarrhea in infants [
66]. We also demonstrated that sufficient diet facilitates more rapid recovery from diarrhea and increase body weight gain in piglets, highlighting the significance of nutritional strategies to moderate HRV infections. On the other hand, the gut microbial diversity did not affect the body weight of HRV challenged pigs, but the HIFM transplantation did significantly decreased the diarrhea severity and duration in both diet groups compared to the GF groups. Zijlstra et al., 1997 and Jacobi et al., 2013, also showed that the quality of the microbiome is an important factor in limiting HRV infection [
38,
50]. These results suggest that the diet might affect the microbiome and host physiology, resulting in alterations in HRV infection and period of morbidity. For example, Zijlstra et al. showed that the decline in the body weight gain and severe diarrhea observed with malnourished piglets challenged with HRV were accompanied by a reduction in villus height and lactase activity, reduced villus:crypt height ratio, reduction in trans-epithelial resistance, and increase in intestinal insulin-like growth factor binding proteins (IGFBP) [
38,
50].
HRV infection in infants was associated with decrease in the gut microbial diversity [
19,
67]; however, in our study, an opposite trend was observed after analysis of intestinal tissues from HIFM+HRV pigs fed with either a sufficient or deficient diet compared to the HIFM+No HRV groups. This finding can be explained by the destruction of the intestinal cells by HRV, which could make more nutrients available for the microbes in the gut [
68]. Furthermore, most of the infants’ studies rely on analysis of fecal samples collected from either mid or late phase of HRV infections [
19,
67]. We also observed that the microbiota quality, not the abundance, in intestinal tissues of the HIFM+HRV pigs was different between the sufficient and deficient diets, suggesting that both HRV infection and the diet may have profound effect on microbial diversity and abundance. As a consequence, the modifications in microbial community caused by the diet could explain in part the reductions observed in clinical signs and bacterial translocation to systemic organs. Both deficient and sufficient diet HIFM+HRV groups displayed unique bacteria present only in one of the diet groups which could serve as biomarkers of HRV infection and may aid in development of novel strategies to moderate HRV diarrhea. For example,
Turicibacter, and
Anoxybacillus were detected only in HIFM+HRV pig intestines. Also,
Turicibacter, Halomonas, and
Shewanella were more abundant in the sufficient diet HIFM+HRV group, suggesting these bacteria could serve as potential bio-indicators of HRV infection and/or host nutrition. Previous association of
Turicibacter species in colon and small intestine of mice was shown to possess immune-modulatory effects through T cells (CD8+) and NK cells activity [
69]. Thus, it is likely that the presence of
Turicibacter species in sufficient HIFM pigs may indicate modulation of immune response promoting recovery from HRV severity.
Unlike the microbiota in intestinal tissues, neither HRV infection nor the diet induced major modifications of the microbiota abundance in the systemic tissues; however, in concordance with impaired intestinal integrity [
50], all systemic tissues of HIFM+HRV groups had a higher microbial diversity compared to the HIFM+No HRV groups, suggesting that HRV infection was associated with a general increase of the microbiota diversity in systemic tissues. Further the diet had an additive effect; however, the increase in diversity was enhanced when pigs were fed deficient diet. These results suggested that HRV infection increases the bacterial translocation to liver, MLN, and spleen likely by compromising the intestinal epithelial barrier; while malnutrition enhances this phenomenon by exacerbating intestinal damage caused by HRV infection [
50].
Though our results clearly demonstrate the interconnections between the diet, microbiota and HRV infection, it should be taken into consideration that only limited number of pigs was used in each treatment group in this study due to the complex nature of experiments with the GF animals. The changes in the gut microbiota in our study may be due to individual or combined effects of the following factors: (i) malnutrition, as malnutrition was shown to affect gut microbiota structure and composition; (ii) HRV pathogenesis- previous studies have shown that enteropathogens including HRV have significant effects on the gut microbiota [
14]; and (iii) the host response or immune response- the host natural defense system are essential for maintaining the homeostasis of the gut microbiota [
62]. Recurrent episodes of diarrhea caused by enteropathogens have a major effect on the gut microbiota [
9]. To substantiate this claim, previous studies have shown that malnourished children, who did not have a diarrheal disease, likely due to enteric infections, did indeed gain weight normally compared to well-nourished children, while the increasing incidence of recurrent diarrhea episodes in malnourished children progressively decreased the weight gain [
70,
71]. Hence, in natural settings, it is clear that the recurrent episodes of diarrhea have the greatest effect on children’s growth likely due to their cumulative effects on gut microbiota with prolonged dysbiosis and intestinal absorptive dysfunction, which is especially problematic in undernourished children [
9].