1. Introduction
Antibiotic-associated diarrhea (AAD) is defined by the presence of diarrhea with three or more soft/liquid stool evacuations within 24 h or an acute and evident change of fecal pattern during antibiotic treatment or also a few weeks later, without other demonstrable causes [
1].
Different mechanisms may determine AAD: a direct toxic effect of antibiotics on the intestine, an altered digestive function secondary to reduced concentrations of gut bacteria, and the over-growth of pathogenic microorganisms [
2]. Mostly, it is a mild condition requiring no treatment, and resolving on its own. More-serious AAD instead needs more appropriate monitoring and specific medications [
2,
3]. An extreme consequence of AAD is the overgrowth of potentially pathogenic organisms, such as
Clostridium difficile [
3].
AAD occurs in about 5–30% of patients either early during antibiotic therapy or up to two months after the end of the treatment [
4,
5,
6].
The frequency of antibiotic-associated diarrhea depends on the antibiotic type (higher risk for penicillins, especially in combination with clavulanate, cephalosporins, and clindamycin), dosage, number of antibiotic prescriptions as well as by host factors (age, prematurity, hospitalization, season) [
7].
In the pediatric population, AAD represents a healthy concern being that the burden and costs are also not well documented by national surveillance studies [
8]. The AAD in patients treated with antibiotics occurred with an average of up to 30%, with a range of 11% to 62% [
9].
Recent trials and metanalysis suggest that specific probiotics are useful in AAD prevention in children [
10,
11].
Goldenberg et al. (2015) performed a systematic literature review, which reported moderate evidence for a protective effect of probiotics in preventing AAD, with an NNT (number needed to treat) of 10. Among the various probiotics evaluated,
Lacticaseibacillus rhamnosus (formerly
Lactobacillus rhamnosus) or
Saccharomyces boulardii at 5–40 billion colony forming units (CFUs)/day appeared to improve NNT [
11].
In a recent review, Guo et al. (2019) added valuable information on AAD prevention by probiotics. In particular, dosage appeared to account for the substantial reduction in the NTT. The authors also reported the subgroup effect based on high dose probiotics (≥5 billion CFUs per day), which revealed an NNT of six [
12]. Overall, these results confirm the importance of strain specificity and dose-related effect of probiotics, as shown by McFarland et al. (2018) [
13].
The present work aims to evaluate, in a primary care setting, the effect of a combination of two microencapsulated probiotics on AAD in a vast cohort of children who underwent antibiotic therapy. The probiotic mixture (patented by Probiotical SpA, Novara, Italy) of
Limosilactobacillus reuteri LRE02–
Lacticaseibacillus rhamnosus LR04 (1.2 × 10
9 CFU of both microorganisms daily, 5 drops per day for 30 days) (Abiflor Baby, Aurora Biofarma R&D) was chosen respecting the new rules recently established by a new European Society of Gastroenterology, Hepatology, and Pediatric Nutrition (ESPGHAN) position paper [
14], i.e., do not contain plasmids carrying antibiotic resistance; bacteria are gastroprotected thanks to the microencapsulation technology; shelf life (i.e., live and viable bacteria on the expiry date of the product) guaranteed for 24 months; the product is “allergen-free”; it does not contain sucrose or fructose.
4. Discussion
The World Health Organization defines probiotics as “live microorganisms which when administered in adequate amounts confer a health benefit on the host” (Food and Agriculture Organization and World Health Organization, 2001). Dr. Metchnikoff opened the “era” of probiotics because he was the first to propose that ingesting certain bacteria could help replace harmful microbes in the body [
17].
As reported in the literature, the minimum quantity sufficient to obtain temporary colonization of intestines by a microbial strain is 10
9 live cells per day [
18]. Therefore, in the case of miscellanea, the recommended daily consumption should contain 10
9 live cells of at least one of the strains, including
Saccharomyces cerevisiae, or lactic acid fermenters, such as
Streptococcus thermophilus or
Lactobacillus bulgaricus [
18,
19].
Lactobacilli, recently reclassified in 23 different genera [
20] and bifidobacteria, are the most common microorganisms used in probiotic preparations. Furthermore, the various products differ in the production processes, which can influence some fundamental characteristics of the probiotic, such as the concentration of viable microorganisms or the presence of contaminants [
19,
21]. The effects of probiotics are strain- and dose-dependent [
1,
13].
The use of probiotics is now widespread both for intestinal and extraintestinal disorders, especially in pediatrics [
3,
22,
23]. A recent statement of the Working Group on Probiotics and Prebiotics of the European Society of Gastroenterology, Hepatology, and Pediatric Nutrition (ESPGHAN) [
1] recommends the use of some probiotic strains for the prevention of AAD. This condition, while transient, is the cause of further morbidity, complications, and sometimes hospitalization with its repercussions in the scope of lost workdays, healthcare costs, and especially baby health [
8].
Children are estimated to use three times more antibiotics than adults [
24] and AAD seems to occur roughly in 25% of children between the initiation of antibiotics and two months after their completion [
25]. Moreover, the use of antibiotics in the first years of life interferes with the microbiota development and results in dysbiosis [
26]. A delicate balance exists between the intestinal microbiota and the host; indeed, all conditions causing an imbalance can potentially result in the occurrence of gastrointestinal or extraintestinal diseases [
27].
It is also noteworthy that dysbiosis related to antibiotic use in the first years of life is a risk factor for obesity [
28], functional gastrointestinal disorders [
29] and impaired neurocognitive outcome [
30].
A Probiotics’ mechanism of action is exerted through the modulation of the content of gut microbiota, maintenance of the integrity of the gut barrier, prevention of bacterial translocation and the modulation of the local immune response by the gut-associated immune system. In particular,
Lacticaseibacillus rhamnosus is a probiotic strain, which when compared to other probiotics, is one of the most appropriate for preventing AAD due to its modest NNT [
31].
Limosilactobacillus reuteri has even been extensively studied in several intestinal conditions, and its therapeutic and preventive effects have been documented (e.g., protection from pathogen colonization, decrease in negative interaction due to environmental stressors) [
31].
In our randomized intervention study, we evaluated the efficacy of a new double-strain probiotic formulation which included
Limosilactobacillus reuteri (formerly
Lactobacillus reuteri LRE02) 2 × 10
8 CFU and
Lacticaseibacillus rhamnosus (formerly
Lactobacillus rhamnosus LR04) 1 × 10
9 CFU on the prevalence of AAD. These probiotic strains are free of plasmids that carry antibiotic resistance, are microencapsulated, and have a shelf life (i.e., live and viable bacteria on the product expiration date) that is guaranteed for 24 months; moreover, in vitro studies have demonstrated a synergistic ability of these two strains when simultaneously cultivated. The product is “allergen-free” [
32] and without sucrose or fructose. Microencapsulation increases the resistance of probiotic microorganisms during the gastro-duodenal transit, thus ensuring their titer and biological activity [
33,
34]. At the same time, it protects cells from degradation phenomena due to external factors (humidity, acidity, osmotic pressure, oxygen, and light) [
33]. One of the advantages using the microencapsulated probiotics is the ability to colonize the intestine in a greater concentration (five times more) compared to non-encapsulated probiotics [
35]. As stated in two different studies by Del Piano et al. (2011 and 2012), the microencapsulation of probiotics offers numerous advantages compared to the freeze-dried technique; the most important of which is the high gut colonization as a result of the increased number of viable cells that transit the intestine and, as a consequence, the reduction in the probiotic concentration delivered in these lipidic microcapsules due to a strong gastro-resistance of the cells [
33,
34].
In our study, the prevalence of AAD was about 50%, regardless of the antibiotic used. This high prevalence, however, previously reported [
7], could be related to the low age of included patients (mean age 28 months). In fact, one of the risk factors for AAD is age < 6 years [
36].
The most prescribed antibiotics, to treat a range of infections reported in
Table 1, were penicillins, followed by cephalosporins and macrolides, according to the recent AIFA (Agenzia italiana del farmaco) National report [
37].
Our results showed a significant reduction in AAD prevalence in group A (probiotic plus antibiotics) (38.5%), compared to the control group B (antibiotics alone) (59.9%).
The type of antibiotics and the reason for their administration do not appear to affect the outcome of the probiotic on AAD, as previously reported for one of two probiotics recommended by ESPGHAN for AAD prevention [
1,
38].
Stool consistency, evaluated with BSS score, was significantly better in group A (mean BSS score 4.5 ± 1.5) than in group B (mean BSS score 4.9 ± 1.6), for all antibiotic categories, both at the end of antibiotic therapy (T0) and after 30 days (T1). In AAD patients, the mean BSS score at T0 was not different between the two groups, regardless of the antibiotic categories used; however, at T1, the BSS score was significantly better in group A than in group B. Surprisingly, in AAD patients, diarrhea started earlier in group A than in group B, regardless of the type of antibiotic used. This effect could be related to the influence of gastrointestinal commensals on motility as previously observed in vivo and ex vivo in mice and rat models, in which the administration of
L. reuteri and different
Lactobacillus species were shown to moderate jejunal motility within minutes [
39,
40,
41]. Another explanation could be the interaction between probiotics and smooth muscle of the intestine as stated by Guarino et al. (2008), which studied an in vitro model of human colonic cells exposed to
L. rhamnosus GG and found a significant shortening of smooth muscle cells that had an impact on motility [
42]. Evidence is accumulating on the hypothesis that certain probiotic strains have acute actions in vivo and ex vivo on the host’s autonomic reflexes and can act differently on the small compared to the large intestine [
40]. These effects can occur within minutes, suggesting that the inter-kingdom signaling responsible for them do not rely on colonization, alteration in the microbiome composition, or any other longer-term adjustments [
43].
However, the number of days with diarrhea was significantly lower in group A than in group B (2.8 ± 1.3 days vs. 3.2 ± 1.4 days), depending on the antibiotic used, as macrolides showed the worst duration. We did not find any difference in the number of stools/day and weight loss in AAD patients in either of the two groups. This is in line with the other studies that have shown that probiotics are associated with a reduction in the mean duration of AAD of 18 h, without having an effect on the number of stools per day [
10,
23].
Our study is not a placebo-controlled study, and a limitation could be the self-reported symptoms made by parents. However, the large number of patients enrolled, and the utilization of the BSS certainly helps to reduce possible biases.
This study has demonstrated that using our probiotic combination from the beginning of antibiotic therapy is also helpful in reducing the mean duration of diarrhea (calculated in days) as well as in obtaining a normal stool consistency in a shorter time.