Pathogen-Specific Benefits of Probiotic and Synbiotic Use in Childhood Acute Gastroenteritis: An Updated Review of the Literature

Probiotics represent viable microorganisms which are found within the normal gut microbiota, that exert strain-specific benefits in the management of several gastrointestinal disorders in children, including acute gastroenteritis. This review aims to evaluate the pathogen-specific role of probiotic supplementation in childhood diarrhea. A search of scientific databases was conducted to identify studies which investigated efficacy of probiotics and synbiotics in influencing outcome of acute gastroenteritis of known etiology. We identified 32 studies, most of which analyzed impact of probiotic supplementation in rotavirus gastroenteritis, while a very limited number of these conducted a separate analysis on bacterial diarrhea. Lactobacillus rhamnosus (L. rhamnosus), L. reuteri and S. boulardii still remain the most researched strains, with a proven role in decreasing diarrhea and hospitalization duration, especially in the setting of rotavirus infection. Combined products containing at least one of the aforementioned strains also performed similarly and might also influence rotavirus fecal shedding. Rotavirus immunization status has also been proposed as a significant influencing factor of probiotic use impact. The paucity of research focusing on bacterial etiologies, as well as of clinical trials conducted within ambulatory care units leaves room for further research on the matter, which needs to include larger cohort studies.


Introduction
Probiotics have been defined as oral supplements which contain viable microorganisms, in the form of bacteria and yeasts similar to those found within the microbiota of the normal, healthy gut [1,2]. The first evidence available in the literature regarding their existence comes from Henry Tissier, who was the first person to remark on a poorer stool bacterial culture in children infected with diarrhea, as well as in those fed with formula, as opposed to healthy and breastfed infants [3,4]. Since the publication of these first data in 1907, many studies have emerged, but their poor design and inadequate cultivation of bacteria in substrates other than human milk initially hindered the acquisition of reliable evidence. However, later research, and the ability to isolate and characterize specific bacterial cultures, showed the numerous health benefits of probiotics, which have been proven to improve intestinal health, alleviate symptoms related to lactose intolerance and decrease the risk of developing disorders such as inflammatory bowel disease, infectious diarrhea or allergies [3,5]. Thus, their administration in adequate amounts can be beneficial for the host and their lack of side effects has encouraged their widespread use [6].

Methods
We searched the Pubmed, Web of Science and Google Scholar databases for randomized controlled trials and pediatric population-based studies which have evaluated efficacy of probiotics and synbiotics in influencing outcome of acute gastroenteritis. We only focused on those studies which included patients with a known infectious etiology of the diarrheic disease or which had also performed a separate analysis on subgroups in which a certain viral or bacterial causative agent was identified or on those cohorts in which rotavirus vaccination status was known. We refrained from reporting data provided by publications limited to abstract formats or by in-extenso manuscripts written in a language different from English. Search terms used included "probiotic" AND "diarrhea" AND "child", or "probiotic" AND "gastroenteritis" AND "child" or "probiotic" AND "rotavirus" or "prebiotic" AND "diarrhea" AND "child", or "prebiotic" AND "gastroenteritis" AND "child" or "prebiotic" AND "rotavirus" or "synbiotic" AND "diarrhea" AND "child", or "synbiotic" AND "gastroenteritis" AND "child" or "synbiotic" AND "rotavirus".

Results
We identified 32 studies which complied with our inclusion and exclusion criteria, as summarized in Tables 1-4. Most of these studies assessed the influence of probiotics on rotaviral diarrhea, whereas a very limited number of clinical trials evaluated the impact of probiotics upon bacterial diarrhea-related symptoms and aftermath. The most intensely researched probiotics on this subject still remain L. rhamnosus, L. reuteri and S. boulardii, but a few others have also showed promising potential in combating the burden of rotaviral or bacterial diarrhea-related hospitalizations and complications. In this paper, we will provide a background on probiotics and synbiotic products and detail how these impact the outcome of childhood acute gastroenteritis with an identified etiology.

Lactobacillus rhamnosus
L. rhamnosus has been proven to be one of the probiotics that can positively influence hospital stay length, as well as diarrhea duration (Table 1) [23]. A European multicenter trial, which randomized an impressive number of children aged 1 month to 3 years of age demonstrated that a combination of oral rehydration and L. rhamnosus is superior to oral rehydration alone in terms of reducing hospitalization duration and number of watery stools after 3 days of diarrhea evolution. Recovery from watery stools was significantly more rapid in those subjects whose stool samples were positive for rotavirus infection. Bacterial etiologies were scarcely detected, not allowing for a convenient statistical analysis, but no differences were reported in terms of diarrhea duration in these cases, independently of the study group allotment [24]. Likewise, a randomized controlled trial conducted in India highlighted the benefits of L. rhamnosus in decreasing diarrhea extent and timespan of parenteral rehydration, but only in those subjects confirmed with rotavirus infection, as opposed to other etiologies [25]. Similar results were reported by Guarino et al., with a slightly more consistent reduction of diarrhea duration in children diagnosed with rotavirus, as well as of fecal viral identification after 6 days of diarrhea onset [26]. Another study, conducted in Taiwan, suggested that L. rhamnosus also aids in decreasing the extent of fecal elimination of rotavirus, in a dose-dependent manner [27]. On the other hand, increase in stool consistency and recovery from diarrheic stools took place after a shorter amount of time with L. rhamnosus administration, but independently of the presence of rotavirus fecal shredding, according to the study of Aggarwal et al. [28]. Moreover, a meta-analysis reported a better performance of L. rhamnosus in children who had not received a vaccine against rotavirus and a lack of significant difference in gastroenteritis outcome in those who were immunized [29]. In terms of bacterial infections, data are scarce; one Indian study in which a little over 60% of the stool cultures were negative, but which individually assessed the role of L. rhamnosus in relation to each particular type of pathogen identified in the stool samples, concluded that its efficacy is limited to reducing diarrhea duration in Clostridium difficile positive patients. However, this conclusion is supported by a very limited number of cases [23].
Two randomized clinical trials which analyzed the efficacy of a combined probiotic strain containing L. rhamnosus and L. helveticus reported no virus-specific benefit over placebo in terms of reducing viral load at 28 days post-enrollment, nor in decreasing intensity of clinical symptoms. Both studies included children diagnosed with gastroenteritis of viral etiologies (norovirus, rotavirus, adenovirus), as well as of bacterial etiologies [21,22].
Still, a decrease in number of diarrhea episodes caused by adenovirus infection was noted (Table 1) [22].
There are limited reports regarding the efficacy of L. rhamnosus in the absence of hospitalization. An Indian study reported a significant reduction in diarrhea episodes during a follow-up period of 4 weeks in recipients of L. rhamnosus diagnosed with rotavirus gastroenteritis, as opposed to those with Crytosporidial diarrhea, in whom administration of the same probiotic showed no benefit compared to placebo. L. rhamnosus also seemed to augment specific IgG (and not IgA) production against rotavirus, but did not influence antibody levels for Cryptosporidium species (Table 1) [30]. Another randomized controlled trial, conducted in Uganda, highlighted the benefit of both L. rhamnosus and S. boulardi in reducing diarrhea episodes only in outpatient settings in children with severe acute malnutrition, and not in those who required hospitalizations. The study suggested that both strains do not influence the outcome and evolution of severe gastroenteritis, but provided no data regarding etiology of the diarrheic manifestations [31].

Saccharomyces boulardii
S. boulardii has been regarded as the most effective agent in reducing duration of diarrhea (including the risk of an evolution of loose stools over 2 days) and is apparently superior to other strains such as B. lactis, L. rhamnosus, L. reuteri and L. paracasei, according to a meta-analysis conducted on 84 studies [33]. Moreover, its advantages over B. clausii in shortening diarrhea extent have been highlighted within a randomized trial, which also showed comparable performance of the two strains in terms of hospitalization, fever duration and number of vomiting episodes [34]. These results contradict former scientific reports such as that of Gaon et al., which claimed that S. boulardii's efficacy is similar to the efficacy of a combination between L. casei and L. acidophilus in diminishing diarrhea and vomiting, when compared to placebo and is independent of Rotaviral etiology (Table 2) [35]. Table 2 details characteristics and results of studies which analyzed overall and pathogen-specific impact of S. boulardii in pediatric gastroenteritis. One randomized controlled trial conducted in India certified the advantages of S. boulardii over a similar product in rotavirus-associated diarrhea, without any adverse events, but only in terms of hospitalization duration. No difference from controls was seen in terms of fever duration, vomiting episodes, proportion of children requiring parenteral rehydration or with diarrhea lasting for more than one week [36]. Similarly, another study conducted by Dalgic et al. established a significant decrease in duration of hospitalization and diarrhea in children diagnosed with rotavirus infection, supplemented with both S. boulardii and zinc, as opposed to controls. Moreover, the authors also demonstrated that this adjuvant therapeutic combination can also accentuate resolution of vomiting [37]. On the other hand, a Bolivian randomized controlled trial reported that number of recurrent vomiting episodes is decreased in patients receiving an oral rehydration therapy in combination with S. boulardii, B. longum, L. rhamnosus and L. acidophilus as opposed to those which only benefited from the addition of S. boulardii in their treatment or who received placebo together with oral rehydration solutions (ORS). Still, S. boulardii alone showed a better outcome than the four-strain product and placebo in shortening the number of days with fever and diarrhea in children infected with rotavirus [38]. Apparently, a three-day treatment with S. boulardii performed even better in inhibiting release of watery stools in pre-school children who tested positive for rotavirus, unlike in those who were negative for the virus [39].
Persistent, prolonged elimination of rotavirus in feces has been reported even in vaccinated children and represents a source of community-acquired infection [40]. S. boulardi might be a potential inhibitor of rotavirus fecal shedding, with absence of fecal virus detection after 5 days of administration, as suggested by a multicenter, case-control clinical trial. This conclusion was, however, supported by a very limited number of cases [41].

Lactobacillus acidophilus
L. acidophilus has been one of the bacteria widely used alone or as part of combined products for the treatment of acute diarrhea in children, in spite of insufficient evidence available [2]. The suppositions surrounding the potential beneficial effects of this strain emerged from evidence supporting its involvement in inhibition of bacterial invasion of epithelial intestinal cells, a consistent characteristic of other Lactobacilli as well [42]. L. acidophilus can also augment specific immune reactions and is stable at high temperatures, so it is unsurprising that several compounds appeared on international markets, which claimed to deliver relief of gastroenteritis-associated symptoms [43]. However, one study and a meta-analysis reported no benefit regarding its complementary use in those pediatric gastroenteritis cases in which an etiological factor was identified from the analyzed stool specimens [44,45]. In similar fashion, one randomized controlled trial reported no clear advantage of L. acidophilus over placebo in cases where rotavirus or norovirus was identified, neither in terms of reducing symptom duration, nor in terms of diminishing stool viral load [46]. Some evidence emerged regarding the advantages of L. acidophilus in shortening hospitalization duration or number of days with fever, but only in patients with unknown etiology of the diarrhea or in whom a rotavirus infection was excluded, as detailed in Table 3 [44,47,48]. Salazar-Lindo et al. also reported a lower number of stools in those children supplemented with L. acidophilus suffering from mild diarrhea, but in their study cohort an infectious agent (namely rotavirus) was identified in only a very limited number of patients [49]. Given the inconsistent results reported by various studies, a meta-analysis conducted by authors belonging to the Working Group on Probiotics and Prebiotics of the ESPGHAN concluded that there is insufficient evidence available to support the administration of L. acidophilus in childhood acute gastroenteritis [50].

Lactobacillus reuteri
L. reuteri strains are able to modulate lipopolysaccharide-induced intestinal inflammation and reduce mucosal damage caused by enteric infections in rats [51]. Both anti-bacterial and anti-inflammatory effects have been associated with their use, as demonstrated in a murine model in which the administration of L. reuteri antagonized the extension of enteral mucosa inflammation and C. difficile infection [52]. Moreover, two in vitro studies also confirmed L. reuteri's potential in combating E. coli growth [53], modulating immune response against Salmonella typhimurium [54] and another study conducted on rats related its use with the development of mucosal rotavirus-specific IgA production [55]. A randomized controlled trial conducted in pre-school children asserted the protective effect of L. reuteri against infectious diarrhea, which lasted through a follow-up period of 3 months after previous administration of the product for the same amount of time (Table 3) [56].
Shornikova et al. reported amelioration of diarrhea in a small pediatric cohort which received L. reuteri, as opposed to placebo, with most of the patients belonging to the intention-to-treat group not experiencing any watery stools after two days of probiotic supplementation. Given that rotavirus infection was identified in 75% of the study population, the authors proposed L. reuteri as a potential therapeutic adjuvant in rotavirus enteritis. However, the study failed to identify any significant increase in specific rotavirus IgA antibody titer in the L. reuteri recipient group [57]. Another research study by these authors, performed on a rotavirus cohort in children aged between 6 and 36 months, confirmed the significant reduction in the duration of watery diarrhea associated with L. reuteri administration, as well as a good restoration of the enteral microbiota in this setting (Table 3) [58].
A Botswanian pediatric randomized controlled trial which included subjects vaccinated against rotavirus found no significant association between the administration of L. reuteri and acute gastroenteritis outcome, whether viral or bacterial. No notable replication inhibition of bacteria such as Shigella, Campylobacter, Enteropathogenic E. coli or Cryptosporidium was reported, but the study did not separately evaluate the subpopulation in which enteric viral infections were found in feces samples, as most of those children also presented another viral/bacterial co-infection [59]. Moreover, a Polish study found no benefit of L. reuteri on diarrhea-related outcomes, regardless of rotavirus vaccination status, but the infectious etiology was unknown in most of the patients enrolled [60].

Lactobacillus plantarum
Lactic acid bacteria have the ability to attach to cellular monolayers, exerting protection against rotavirus, as well as against the transmissible gastroenteritis virus (TGEV), according to Maragkoudakis et al. [61]. Out of all the Lactobacillus species studied, L. plantarum had the highest attachment ability and was found to have an antiviral effect against both viruses, as proven by the same experimental study [61]. Another study performed on mice proved that exopolysaccharides released by L. plantarum effectively reduce the number of diarrheic episodes and rotaviral shedding [62]. The decrease in rotavirus load in subjects supplemented with a L. plantarum product was later confirmed by research conducted in children, which also showed a significant decrease in diarrhea duration and in its severity (assessed through daily changes in Vesikari score- Table 3 [32]) [63].   Table 4 provides insights regarding the specific beneficial use of combined probiotic products in childhood diarrhea. A double-blind randomized controlled trial reported effective reduction of diarrhea duration in children who were administered twice daily a combined suspension, consisting of 6 different strains: B. longum, B. lactis, L. acidophilus, L. rhamnosus, L. plantarum and Pediococcus pentosaceus. The study also established that each individual component of the combined aforementioned suspension can inhibit in vitro rotavirus infection, without cytotoxic effects on the population of cells analyzed [64]. A B. longum and L. acidophilus mixture can also significantly shorten diarrhea duration, according to a Korean study [65]. Another similar study demonstrated significant reduction in hospitalization duration in children infected with rotavirus who received a mixed probiotic preparation consisting of L. acidophilus, L. rhamnosus, B. longum and S. boulardii for at least 5 days as opposed to oral/parenteral rehydration alone [66]. A preparation containing Bacillus mesentericus, Clostridium butyricum and Enterococcus faecalis also seems to effectively decrease diarrhea severity in combination with symptomatic and rehydration therapy after 3 days of daily two-dose administration in patients infected with rotavirus, but not in those infected with Salmonella [67]. Similarly, the administration of a suspension containing L. casei, L. rhamnosus, Streptococcus thermophilus (S. thermophilus), B. breve, L. acidophilus, L. bulgaricus and B. infantis twice daily for a minimum of 5 days reduced the number of hospitalization days related to rotaviral diarrhea [68]. A combination of 8 probiotic strains (L. acidophilus, L. paracasei, L. bulgaricus, L. plantarum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum and S. thermophilus) has also been proposed as an adjuvant therapeutic agent in rotavirus diarrhea, as it promoted a shorter recovery time and significantly decreased the requirement of oral rehydration therapy and stool volume loss after just two days of administration [69]. Furthermore, the simultaneous administration of L. rhamnosus and L. reuteri supposedly reduces the detection rate of rotavirus fecal antigen after 5 days of treatment, which suggests that these two probiotics positively interfere with viral elimination time. Still, this evidence is supported by a study with very limited follow-up time [70].

Synbiotic Products
Prebiotic supplements in the form of pectin-derived acidic oligosaccharides, shortchain galacto-oligosaccharides, long-chain oligosaccharides added to a fermented milk concentrate have been proven to enhance protection against rotavirus infection in suckling rats, through a presumed interaction with viral particle attachment to the enterocyte, which consequently inhibits viral replication [71,72]. Synbiotic products, containing both prebiotics and probiotics seem to also stimulate immune response against enteric viruses through modulation of the interferon signaling pathway [73]. Hence, synbiotics have been proposed as additional products to classical therapy in severe rotavirus enteritis by a review mainly based on in vitro studies [74]. The authors acknowledged, however, that the paucity of randomized controlled trials mandated future research investigating the preventive and therapeutic benefits of synbiotics in rotavirus diarrhea [74].
Evaluation of etiology-based performance of synbiotics in the management of diarrhea in children has been so far based on two randomized-controlled trials, one of them including only patients in whom stool samples were positive for rotavirus and the other one conducting a separate analysis on children with diarrhea triggered by the same etiological agent [75,76]. As detailed in Table 4 lactis together with inulin were administered, as opposed to children infected with adenovirus or Entamoeba histolytica, in whom the synbiotic produced no significant difference [75,76]. Another three studies have also supported products consisting of multiple probiotic strains and FOS as complementary agents to standard treatment that can yield a shorter recovery of stool consistency and overall decrease in duration of diarrhea [77][78][79]. Moreover, L. paracasei combined with arabinogalactan and xylo-oligosaccharides can improve stool output frequency, positively impact hospital stay and decrease parental working days absences, according to an Australian study [80]. However, all these four studies have provided no information regarding diarrhea etiology for any of the pediatric patients enrolled [77][78][79][80]. Table 4. Characteristics of clinical studies which assessed overall and pathogen specific impact of combined probiotic strains and synbiotic supplementation in childhood diarrhea.

Discussion
The latest position paper of the Working Group on Probiotics and Prebiotics of the ESPGHAN, published in 2022, provides weak recommendations for the use of L. rhamnosus and S. boulardii in the management of acute pediatric gastroenteritis, based on low evidence [68]. An even lower level of evidence sustained the administration of L. reuteri for the same purpose, whereas a recommendation was issued against the use of Bacillus clausii and against the combination of L. reuteri and L. helveticus in childhood diarrhea, similarly to their previous publication [18,81]. The authors have provided no specific advice for diarrhea with an established etiology and have also mentioned the potential impact of rotavirus vaccine coverage in influencing their analysis of previously published data [81].
This review tried to add novelty to the existing literature data by assessing the impact of probiotic and synbiotic use upon symptom duration, severity and immune response in both viral and bacterial gastroenteritis in children. Most of the studies conducted so far which assessed the impact of probiotic use upon childhood enteric infections have focused solely on rotavirus diarrhea and provided the basis for two previous meta-analyses [82,83], as rotavirus still remains the leading cause of diarrhea-associated complications, especially in underdeveloped countries [84,85]. However, norovirus seems to overcome rotavirus as the etiological diarrheic agent which most commonly requires medical care in children, in those countries with high anti-rotavirus vaccination coverage, such as the United States [86]. Several theories have emerged regarding the possible inhibition of replication exerted by probiotics upon enteric viruses, and probiotic supplementation has been recommended for prevention of rotaviral diarrhea, as well as for diminishing symptom duration [82,85]. As previously presented and detailed in Tables 1-4, several strains have proven their benefits in rotavirus-associated diarrhea, including individual strains, as well as combined probiotic preparations. The majority of these studies involved supplementation with either L. rhamnosus or S. boulardii, and combined suspensions included at least one of these two strains, which still represent the most studied and validated probiotics in the pediatric literature [18,87]. One study which analyzed a combined probiotic product was omitted from Table 4, due to the lack of information regarding the exact content of the product used, as only a commercial name was provided in the manuscript abstract and main text [88]. However, very few studies separately analyzed the impact of probiotic use upon recovery from bacterial diarrhea [23,30,59]. This might be explained by the small study populations included within randomized controlled trials, and the even more size-limited sub-cohorts in which a bacterial etiology was identified. Experimental research studies have suggested that probiotics such as S. boulardii and several Lactobacillus strains show antimicrobial effects, but with limited sensitivity against the analyzed pathogens [89,90].
On the other hand, several studies have evaluated the impact of probiotic strains such as L. reuteri or of a product consisting of B. lactis, L. rhamnosus, and L. acidophilus on diarrhea severity and duration, in spite of not reporting any information regarding the source of infection, which might have greatly influenced the analyzed outcome parameters [91][92][93]. In similar fashion, one Indian study asserted that S. boulardii reduces time until resolution of watery stools and duration of diarrhea after 5 days of treatment, but conducted no separate etiology-based evaluation, due to the very limited number of cases in which the source of diarrhea (rotavirus/Vibrio cholerae) was known [94]. Synbiotics containing FOS or inulin might also positively influence childhood gastroenteritis outcome, but there is insufficient data to assess the true impact of their use in particular infectious settings [95,96]. Consequently, the most recently published Cochrane systematic review underlines that there is insufficient data to allow pathogen-specific analysis of the role of probiotic supplementation in acute gastroenteritis, due to the paucity of the studies which have identified etiological agents of diarrhea or have conducted a separate statistical evaluation, focused on a certain pathogen [97]. Furthermore, the question of adequate, safety/efficacy balanced dosage of even the most widely used strains still remains into place and experts in the field suggest conducting trials comparing administration of different dosages of the same/various strains [98].
It is also worth mentioning that most of the studies conducted so far on the matter of probiotic efficacy in acute pediatric gastroenteritis included hospitalized children, and a very limited number of them were performed in outpatient settings or included follow-up of patients' outcome after discharge from the hospital [30,31]. A decrease in the duration of diarrhea in relation to L. reuteri use was reported within a Turkish study performed in an ambulatory care unit [92]. Still, as represented in Tables 1-4, most of the studies which evaluated pathogen-particular benefits of probiotics have only evaluated their efficacy after 5-7 days of administration.
Recent literature data supports the administration of probiotics also as a prophylactic measure against several enteric infections. Two studies conducted on a murine model showed that S. boulardii can effectively inhibit tissue inflammation and bacterial translocation, which provide protection against Salmonella [99,100]. Prevention of rotavirus gastroenteritis seems on the other hand to be enhanced by L. rhamnosus administration, especially in hospitalized children [101], as well as by supplemented formula feeding, containing strains such as Bifidobacterium animalis subspecies lactis BB12 alone/in combination with S. thermophilus [102]. L. acidophilus also enhanced protection against rotavirus, according to a meta-analysis conducted by Di et al. [103]. L. reuteri on the other hand has been regarded as a possible prophylactic agent against diarrhea in pre-school children [104], helping maintain the integrity of the intestinal mucosa epithelial barrier, offering protection against its disruption, often caused by enterotoxigenic E. coli infection [105]. Moreover, L. plantarum interferes with binding of enteropathogenic E. coli to the intestinal epithelial cells [106]. The results obtained among in vitro studies still require confirmation among large-cohort studies and as Depoorter et al. suggest in their review, there is overall insufficient proof to routinely recommend probiotics for prevention of acute gastroenteritis in children [107].

Conclusions
Rotavirus infection remains the most frequently evaluated etiology in relation to probiotic efficacy, with multiple studies proposing a decrease in its related diarrhea and hospitalization duration with widely used probiotics such as S. boulardii, L. reuteri and L. rhamnosus, as well as synbiotic products containing at least one of these three strains, as complementary treatment to supportive therapy. There is, however, insufficient data on the impact of probiotics in relation to rotavirus immunization status or their influence on the diarrhea episodes treated in outpatient settings. Clinical reports are scarce regarding potential probiotic benefit in bacterial diarrhea, but some strains have been shown to inhibit in vitro growth of certain microbial pathogens. Future studies, conducted on larger cohorts, are warranted to assess the individual and additive effects of probiotic strains on outcome of pediatric gastroenteritis with identified causative agents.