Effects of Probiotics Supplementation on Risk and Severity of Infections in Athletes: A Systematic Review

The aim of this review was to appraise the literature on the effects of probiotics supplementation on gastrointestinal (GI) and upper respiratory tract infection (URTI) risk and prognosis in athletes. The search was conducted using the following electronic databases: MEDLINE (PubMed); Web of Science; Scopus; and SPORTDiscus (EBSCO). According to the PRISMA guidelines, randomized controlled studies performed on healthy athletes with a note dose of probiotics supplementation were considered. From the 2304 articles found, after eliminating reviews and studies on animals and unhealthy subjects and after screening of titles and abstracts, 403 studies were considered eligible. From these, in accordance with the inclusion and exclusion criteria, 16 studies were selected, ten of which concerned endurance athletes. The majority of the studies reported beneficial effects of probiotics in reducing the risk of developing the examined infections or the severity of related symptoms. However, due to the differences in formulations used and populations analyzed in the available studies, further research is needed in this field to achieve stronger and more specific evidence.


Introduction
Nowadays, it is well established that physical activity (PA) can have beneficial effects on the whole human body and strength its immune defenses. In fact, scientific evidence has established that regular PA is effective in the prevention of various chronic diseases, such as cardiovascular diseases, diabetes, cancer, hypertension, obesity, depression, osteoporosis, and premature death [1,2]. However, the effects of PA can be different depending on its intensity and duration.
In particular, high-intensity PA and strenuous exercise seem to increase intestinal permeability and diminish gut mucus thickness, potentially enabling pathogens/toxins to enter the bloodstream, and have been associated with immunosuppression by decreasing immune cell function, which enhances susceptibility to infections [3][4][5][6][7][8][9].
In fact, during heavy training and competitions, a higher incidence of gastrointestinal (GI) disorders, such as diarrhea and heartburn [7,8], and upper respiratory tract infections (URTIs) can occur [9][10][11]. This is due to acute post-exercise immune breakdowns and chronic suppression of immune factors, dependent on frequent strenuous exercise [12,13]. Therefore, reducing these symptoms in athletes becomes a top priority. Nowadays, probiotic supplement use is promoted in this perspective. The term "probiotic" refers to those microorganisms, which, once ingested in adequate quantities, are able to exercise beneficial functions for the body [14].
Indeed, there is increasing evidence to support the efficacy of probiotic supplementation, alone or in combination with prebiotics, in reducing the number, duration, and severity of acute infectious diarrhea and URTI cases in the general population [15][16][17]. The interest in athletes' diet supplementation arises from this evidence. Essentially, probiotics would act by moderating the negative effects of acute and chronic intense exercise on the immune system, thus reducing susceptibility to the aforementioned pathologies [15][16][17].
The main aim of this review was to critically appraise the literature on the effects of probiotics supplementation in the prevention of GI and URTI in athletes. The role of probiotics in the reduction of GI and URTI related symptoms was also evaluated.

Materials and Methods
This systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [18], according to a protocol published in the PROSPERO database (CRD42021268105).

Eligibility Criteria
Following the PICO model for a systematic review, we selected the studies considering these parameters: patients (P), healthy adult athletes; intervention (I), note dose of prebiotics supplementation; comparison (C), comparison with a placebo group; outcomes (O), prevention of gastrointestinal and respiratory tract infections. Only randomized controlled studies performed on healthy adult athletes with a note dose of probiotics supplementation were included. The inclusion and exclusion criteria adopted for the systematic review are shown in Table 1.

Outcomes
We considered as outcomes the effects of probiotics supplementation on the prevention of gastrointestinal and respiratory tract infections in athletes. Where no changes in the incidence of the aforementioned infections were reported, the effects of probiotics on the severity of symptoms were considered.

Literature Search and Selection of Studies
The search was conducted until 27 July 2021 using the following terms: ("probiotic") AND ("sport" OR "exercise" OR "athlete" OR "physical activity"). We searched the following electronic databases: (1) MEDLINE (PubMed); (2) Web of Science; (3) Scopus; and 4) SPORTDiscus (EBSCO). Moreover, we manually searched for further articles in the reference lists of available reviews. We did not restrict the search by date or publication status. Only articles in English, Spanish, Italian, and French languages were included.

Data Collection
Two reviewers (M.D.D. and P.C.) independently assessed the results of the electronic search. Duplicate articles were excluded, then potentially eligible studies were identified and selected based on title and abstract. Full texts of the potentially eligible studies were thoroughly reviewed independently by the same reviewers and those studies that met the selection criteria were included in the analysis. Disagreements were resolved by the opinion of another reviewer (V.D.O.).
For each study, the following information was tabulated by another two reviewers (G.C. and C.P.P.): author and year of publication, main aim of the study, sample size and demographic characteristics, type of study design, type, dose, administration route and length of the supplementation, and main results on health and performance.

Risk of Bias
The risk of bias of the studies was assessed using the revised Cochrane Risk-of-Bias tool for randomized trials (RoB2) [19]. The evaluation was performed by two independent reviewers (P.C. and G.C.). Disagreements were resolved by a third reviewer (M.D.D.). The randomization process, deviations from intended interventions, data on missing outcomes, measurement of outcomes, and selection of reported outcomes were reviewed. Based on this assessment, the risk of bias for each study was categorized as low, moderate, or high.

Study Selection and Characteristics
As shown in Figure 1, the primary search identified 2304 relevant articles. After eliminating studies on animal and on unhealthy subjects and reviews, and after the screening of titles and abstracts, 403 studies were considered eligible. From these, duplicates and non-randomized studies were then eliminated and, in accordance with the inclusion and exclusion criteria (  The selected studies were published between 2006 and 2021, and they were carried out in different geographic areas: Europe (Austria, UK, Leicestershire, Finland, Serbia), West Asia (Israel), East Asia (Japan), South America (Brazil), and Oceania (New Zealand). All the studies were randomized and the majority of them had a double-blind, placebocontrolled design. Table 2 shows the sample characteristics for each study. A total of 915 subjects were included and were considered in the analyses of the present review; four studies did not report the participants' gender [23,24,31,33], seven studies involved only males [20][21][22]26,28,31,34], and five studies included both genders [25,27,29,30,35]. The studies involved adult participants with an average age between 19 and 50 years. The selected studies were published between 2006 and 2021, and they were carried out in different geographic areas: Europe (Austria, UK, Leicestershire, Finland, Serbia), West Asia (Israel), East Asia (Japan), South America (Brazil), and Oceania (New Zealand). All the studies were randomized and the majority of them had a double-blind, placebo-controlled design. Table 2 shows the sample characteristics for each study. A total of 915 subjects were included and were considered in the analyses of the present review; four studies did not report the participants' gender [23,24,31,33], seven studies involved only males [20][21][22]26,28,31,34], and five studies included both genders [25,27,29,30,35]. The studies involved adult participants with an average age between 19 and 50 years. The total number of CD8 T cells and the memory subsets statistically decreased only in the placebo group post-race. Pro-inflammatory cytokine production by stimulated lymphocytes decreased in the probiotic group after the supplementation period. Data from the URTI questionnaire showed that both placebo and probiotic group presented low level of URTI with no statistical difference between the groups. Regular ingestion of L. salivarius does not appear to be beneficial in reducing the frequency of URTI in an athletic cohort and does not affect blood leukocyte counts or levels of salivary antimicrobial proteins during a spring period of training and competition. Drink contained a minimum of 6.5 × 10 9 live cells of L. casei Shirota (twice per day) 5 months A significant time × group interaction effect was observed for plasma CMV antibody titers in CMV seropositive participants (p < 0.01) with antibody titer falling in the PRO group but remaining unchanged in the PLA group over time. A similar effect was found for plasma EBV antibody titers in EBV seropositive participants (p < 0.01) with antibody titer falling in the PRO group but increasing in the PLA group over time.
In summary, regular ingestion of PRO did not reduce URS episode incidence which might be attributable to the low URS incidence in this study. Regular ingestion of PRO reduced plasma CMV and EBV antibody titers, an effect that can be interpreted as a benefit to overall immune status. LGG had no effect on the incidence of respiratory infections or GI-symptom episodes in marathon runners, but it seemed to shorten the duration of GI-symptom episodes. CD86 as maturation marker on pDC was significantly increased in the LC-Plasma group. Cumulative days of URTI were significantly lower in the LC-Plasma group and symptoms such as sneezing or running nose were significantly lower in the LC-Plasma group. Moreover, the cumulative days of fatigue were significantly fewer in the LC-Plasma group.  There was a substantial 0.7 (0.2 to 1.2) scale step reduction in the severity of gastrointestinal illness at the mean training load in males, which became more pronounced as training load increased. The load (duration × severity) of lower respiratory illness symptoms was lower by a factor of 0.31 (99%CI; 0.07 to 0.96) in males taking the probiotic compared with placebo but increased by a factor of 2.2 (0.41 to 27) in females.
Regarding the risk of bias assessment, eleven studies showed a low risk of bias (Table 3).

Single-Strain Probiotics Supplementation Prevention of Respiratory Infections
Eight studies investigated the effects of single bacterial strain probiotic supplementation on the prevention, occurrence, duration, and severity of respiratory tract infections. Cox et al. conducted a trial on a group of healthy elite male distance runners for a duration of 28 days. The supplementation of a probiotic composed of Lactobacillus fermentum VRI-003 resulted in a reduction in the number of days of respiratory infection and a tendency to a lesser extent of the disease [21]. Gill et al. evaluated the effects of a short-term (7 days) supplementation of L. casei on a group of healthy endurance-trained male runners. They deduced that this kind of supplementation does not provide further immune protection at the level of the respiratory oral mucosa [22]. Gleeson et al. conducted two studies in which they used two different bacterial strains for 16 weeks. In the first study, they administered a probiotic based on L. casei shirota to a group of healthy subjects who were engaged in regular sports training (various sports); in this case, there was a reduction in the frequency of respiratory infections correlated with a better maintenance of salivary immunoglobulin A (IgA) levels [23]. In the second study, they conducted the experimentation on a group of endurance athletes by administering them a probiotic based on L. salivaris. In this case, there was no reduction in the frequency of respiratory infections, nor did the supplementation affect lymphocyte counts or antimicrobial protein levels [24]. In a further study, Gleeson et al. evaluated the effects of long-term (5 months) supplementation of a probiotic based on L. casei Shirota on a group of endurance athletes. This kind of supplementation did not reduce the episodes of respiratory infections, however, it resulted in a reduction in antibodies towards cytomegalovirus and Epstein-Barr virus in people with these infections [25]. Komano et al. found a reduction in cumulative days of respiratory infection symptoms following the short-term (13 days) administration of a probiotic based on Lactococcus lactis to a group of healthy male athletes [28]. Marinckovic et al. found only a trend towards a reduction in the severity of respiratory infections, the duration of episodes, and the number of symptoms (L. helveticus Lafti L10) [29]. However, West et al. obtained uncertain results following the administration of L. fermentum VRI-003 PCC for 11 weeks in a group of agonist cyclists. The burden (duration × severity) of lower respiratory disease symptoms was lower only in males, while it increased in females [35].

Prevention of Gastrointestinal Infections
Regarding the possible effects of probiotic supplementation on the prevention of and reduction in the incidence of infections affecting the gastrointestinal tract, two studies have investigated this. Kekkonen et al. found a reduction in the duration of episodes of GI infections following the supplementation of L. rhamnosus for a period of 3 months to a group of marathon runners [27]. West et al. found a decrease in the severity of GI disease by administering a probiotic based on L. fermentum VRI-002 PCC to a group of agonist cyclists for a period of 11 weeks [35].

Discussion
The studies included in this review concern experiments conducted from 2006 to 2021 examining the effects of probiotics on athletes in the context of various sports disciplines.
In particular, these studies aimed to investigate how the use of probiotics can prevent or alleviate the main infections related to high-intensity exercise.
With regard to disorders affecting the GI tract, the results suggest that both the integration of single-strain probiotics, based mainly on Lactobacillus spp., and that of multispecies probiotics produce protective effects against these pathologies, after a longterm administration ranging from a minimum of 4 weeks to a maximum of 27 weeks. With regard to the integration of single-strain probiotics, minimal dosages, compared to the higher ones experienced in the administration of multispecies probiotics, but taken on a long-term basis, are already able to help in reducing the duration of GI infections. However, the use of probiotics based on multispecies probiotics at even lower dosages and fewer times can provide more effective protection. In particular, the association of Bifidobacteria, Lactobacilli, and E. faecium is able to reduce the incidence of nausea, belching, and vomiting and of GI symptoms due to intense physical exercise.S Comparing the effects of single bacterial strains with that of multispecies probiotics, it can be deduced that while the former can reduce the duration of episodes of disease/disorders and the severity of infections, the latter actually have a predominantly preventive action, managing to intervene in intestinal permeability and reducing the incidence, as well as the severity, of symptoms affecting the GI tract.
As for respiratory diseases, the results are encouraging in the case of both single-strain and multispecies probiotics consumption. In particular, the administration of L. casei Shirota has been shown to be effective in reducing the frequency of respiratory infections. Shortterm administration of L. lactis at high doses also resulted in a reduction in cumulative days of respiratory symptoms. L. fermentum VRI-003 led to a reduction in the number of days of illness and a tendency to a reduction in the severity of the disease (administration for 28 days, 2 billion), and a reduced symptom burden (given for 11 weeks, 1 billion). On the other hand, no results have been obtained from the administration of L. salivarius, albeit in high doses and for a long period, of L. casei after a short-term administration (7 days), and of L. casei Shirota in another study, which, however, found a low incidence of infections.
With regard to the effects of multispecies probiotics, the combination of various strains of Lactobacilli and Bifidobacteria has determined excellent results in terms of reduction in incidence, further underlining the power of multispecies integration. Only one study did not show any results, which is probably attributable to the poor variety of species administered in the 30 days before the competition. In fact, in another considered study, which provided a lower dose of probiotics (5 billion) and a richer variety of species for an equal time, a mitigation of the incidence of infections was found. So, contrarily to the theory that the use of each bacterium separately can produce results while their combination can be less effective or not at all effective [36], in this case it is precisely the heterogeneity of the probiotics that gave a greater effectiveness.
As regards the type of physical exercise, the most represented one is endurance sports [20][21][22]24,25,27,30,32,34,35] in which the supplementation of probiotics has determined, in most cases, positive effects in terms of mitigation of the symptoms associated with respiratory and gastrointestinal tract infections and, in one study, in terms of reducing the incidence of respiratory [34] infections. In this case, the supplementation concerned multispecies probiotics, further underlining the importance of the association of multiple bacterial species.
While there are only two studies [26,31] involving rugby players in the review, they offer interesting results. In both studies, supplementation included the use of multispecies probiotics following which a reduction in the incidence of gastrointestinal and respiratory infections was recorded.
As for the studies conducted on subjects who performed various types of physical exercise [23,28,29,33], the supplementation with a probiotic based on L. casei Shirota produced a reduction in the frequency of URTIs in one study [23]. Furthermore, the supplementation of multispecies probiotics in this case also determined beneficial effects in terms of reducing the incidence of URTI.
As for the formulations used for the administration of probiotics, in most of the studies including probiotics these were administered in capsules including maltodextrin as an additive. Maltodextrin is a polysaccharide, used as a food additive and commonly added as a stabilizer, coating material, or bulking agent. Scientific studies conducted in recent years have concerned the potential effects of the consumption of maltodextrin, not only in animals, but also in humans. In particular, it was shown that in humans the use of maltodextrin represents a stressor for the endoplasmic reticulum of intestinal epithelial cells with a consequent reduction in mucus production downstream, thus determining an increase in susceptibility to colitis [37]. In the case of athletic subjects, intense physical activity causes an increase in intestinal permeability, which makes the intestinal barrier more susceptible to the entry of substances such as toxins and pathogens. So, it should be clarified whether the maltodextrin used in probiotic supplements can somehow negatively affect the beneficial effects of the probiotic itself. All this must also be contextualized with respect to the type of diet followed and, therefore, the overall quantity of maltodextrin taken in the diet.
This systematic review has strengths and limitations. The strengths concern the level of evidence coming from the analyzed studies, which are all randomized and showed a generally high quality. As for the limitations, in the included research, probiotics are administered in different quantities and forms for a variable duration of the intervention. In particular, as underlined in the review, some studies were based on the administration of a single-species probiotic, while others examined the effects of multispecies compounds, which led us to separate the corresponding results. Furthermore, some of the included studies did not use probiotics alone but with other substances, making it difficult to interpret the results.
It is difficult to conceive of a probiotic as primary prevention, and it will probably never be possible to consider its real benefit in secondary prevention. Future research could be useful, for example, in the context of network meta-analyses to analyze the best probiotic formulation, but it is still difficult, not being able to build a reliable control group. Probiotic supplementation has traditionally focused on gut health, however, performance increases that have been underestimated in recent years should be further investigated in the context of possible doping effects [38,39].

Conclusions
In recent years, the interest in the effects of probiotics on athletes' health has been growing worldwide. In particular, the evidence coming from the available literature suggests that the integration of the athletes' diet with certain bacterial strains could be useful to help them to prevent infections affecting the gastrointestinal and respiratory systems, or to reduce the related symptomatology. Since intense physical exercise can increase the risk of developing these infections by lowering the immune defenses of athletes, the perspective of probiotic use in a sport setting is worthy of attention. In order to achieve stronger and more specific evidence on this issue, further research is needed to identify which category of athletes can benefit most by probiotics supplementation, which microbial species can be more effective, and in which circumstance (i.e., climatic conditions, sport season, epidemiological situations, etc.) their use can be recommended.