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Probiotics for the Prevention of Antibiotic-associated Diarrhea in Adults

Objective: This meta-analysis aims to combine the latest research evidence to assess the effect of probiotics on preventing antibiotic-associated diarrhea (AAD) in adults. Methods: PubMed, Cochrane Library, EMBASE, and Web of Science were searched for randomized placebo-controlled trials on probiotics preventing AAD. A random or fixed effect model was used to combine the incidence of AAD (primary outcome) and the adverse event rates. The authors performed subgroup analyses to explore the effects of different participants population, probiotics species, and dosage. Results: Thirty-six studies were included with 9312 participants. Probiotics reduced the incidence of AAD by 38% (pooled relative risk, 0.62; 95% confidence interval, 0.51-0.74). The protective effect of probiotics was still significant when grouped by reasons for antibiotics treatment, probiotic duration, probiotic dosage, and time from antibiotic to probiotic. However, there were no statistically significant increased adverse events in the probiotics group (relative risk, 1.00; 95% confidence interval, 0.87-1.14). Conclusions: This updated meta-analysis suggested that using probiotics as early as possible during antibiotic therapy has a positive and safe effect on preventing AAD in adults. Further studies should focus on the optimal dosage and duration of probiotics to develop a specific recommendation.

A ntibiotic-associated diarrhea (AAD) is defined as diarrhea developing from the beginning of antibiotic treatment to 6 to 8 weeks after discontinuation, which may contribute to antimicrobial prescription noncompliance and the overconsumption of second-line antibiotics. 1 The prevalence of AAD varies between 5% and 39% in adults. It largely depends on the antibacterial spectrum and pharmacokinetic characteristics including the absorption rate of oral administration and enterohepatic circulation of parenteral administration. 2 The pathogenesis of AAD includes the following 2 aspects: (1) the direct effect of antibacterial agents on the intestinal mucosa; (2) the interference of antibacterial agents on the intestinal flora ecosystem, which leads to normal metabolic dysfunction and overgrowth of pathogens (especially Clostridioides difficile). 3 As a live microorganism, probiotic with adequate amounts can bring health benefits to the host. 4 The mechanisms by which probiotics work on AAD may associate with the following: (1) altering the gut microbiota composition and metabolism; (2) modulating the solute secretion and absorption; and (3) improving the intestinal barrier function and intestinal immune responses. 5 Although several randomized controlled trials (RCTs) and meta-analyses have shown its efficacy in preventing AAD, there are currently no clear clinical practice guidelines for probiotics use in preventing AAD. 6 A review comparing the effectiveness of multiple probiotics suggested that positive or negative generalization about probiotics was inadequate. Strain specificity, the designated patient population, and various treatment conditions would change the effect of probiotics. 7 Therefore, our meta-analysis aims to combine the latest research evidence and compare the effects of probiotic

METHODS
This meta-analysis was conducted strictly following the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses. 8

Selection Criteria
Inclusion criteria: (1) patients limited to the adults both inpatients and outpatients who were prescribed antibiotics for various reasons with probiotics (experimental groups) or placebo (control group); (2) providing the occurrence of AAD; and (3) the study designed as an RCT.

Literature Search
The databases involving the PubMed, EMBASE, Web of Science, and Cochrane Library were searched for the RCTs on probiotics to prevent AAD. Publications in any language from the databases inception to February 2020 were included. The search terms were the combinations of the following Mesh terms and key words: "probiotic(s)," "diarrhea," "anti-bacterial agents," "antibiotic(s)," "antibiotic-associated diarrhea," "placebo," "randomized," and "randomized controlled trial."

Data Extraction and Quality Assessment
The data extraction was conducted using the standardized form by 2 independent researchers (W.L. and Q.Z.). The primary outcome was the occurrence of AAD during the follow-up period. The secondary outcome was the incidence of adverse events. Other data extracted included demographics, participant setting, indications for antibiotics, probiotics species and dosage, probiotics duration, time from antibiotics to probiotics, follow-up period, and diarrhea definition.
The Cochrane Handbook for Systematic Reviews of Interventions 9 was applied to assess the quality of the selected studies. Two researchers assessed the eligibility and quality of each article independently. Any discrepancies were resolved through consensus, adjudicated with the support of a third investigator.

Statistical Analyses
We used the RevMan V.5.2 10 and Stata Release V.15.1 (StataCorp, College Station, TX) to perform the data analyses. The pooled relative risk (RR) and the 95% confidence interval (CI) were determined by a random-effects model (DerSimonian-Laird method 11 ) or a fixed-effects model (Mantel-Haenszel method 12 ). The χ 2 test and I 2 statistic were used to evaluate the heterogeneity of included studies. 13,14 P < 0.1 or I 2 > 50% indicated substantial heterogeneity and a random effect would be adopted. Otherwise, a fixed-effects model would be applied. Sensitivity analysis and subgroup analyses were carried out to explore the sources of heterogeneity. In addition, we assessed the publication bias by the funnel plot, Begg test, and Egger tests. [15][16][17] Figure 1. The probiotics species studied in the trials primarily included Lactobacillus, Saccharomyces, Bifidobacterium, and Streptococcus. Probiotics were used at the same time as antibiotics or were prolonged by 2 to 28 days after the therapy. Diarrhea was defined by the World Health Organization (WHO) criterion in 8 studies ( ≥ 3 loose stools within a 24-h period). 54 Six studies applied an adjusted WHO criterion ( ≥ 3 loose or liquid stools/day for at least 2 d). Other RCTs defined diarrhea based on the number of bowel movements per day and the consistency of the stool. Table 1 summarizes the details of participants and intervention.

Quality Assessment
The quality assessment results are shown in Figure 2

Sensitivity Analyses and Subgroup Analyses
Sensitivity analysis revealed that the pooled RR of probiotic effectiveness was robust. No single study significantly affected the overall effect.
Based on the characteristics of the studies, such as the quality of publications, age, participant setting, dosage, and intervention duration, we carried out a series of subgroup analyses. There were significant differences (P＜0.1) among the 4 subgroups including reasons for antibiotics treatment (P = 0.0007), probiotic duration (P = 0.006), probiotic dosage (P = 0.05), and time from antibiotic to probiotic (P = 0.03).
Other subgroups, as shown in Table 2, were also evaluated but were not statistically different.

Adverse Events
A total of 15 studies described adverse events, mainly involving nausea, bloating, and dyspepsia. Four of them reported no adverse events either in the probiotics group or in the placebo, and 2 registered serious adverse events but not attributable to probiotics. There were no statistically significant increased adverse events in the probiotics group (RR, 1.00; 95% CI, 0.87-1.14; P = 0.97 ) (Fig. 5).

Publication Bias
The funnel plot, Begg test, and Egger test were applied to assess the publication bias of the enrolled studies. These results provided evidence of publication bias (Begg test: z = 2.36, Pr > |z| = 0.018 < 0.05; and Egger test: t = −4.77; 95% CI, −2.40 to −0.97; P < 0.05). We use the trim and fill method to correct the publication bias and yielded the same pooled RR of 0.62 as initial outcomes, which suggested that results of the overall effect were stable, and publication bias had few effects on the results. Therefore, our asymmetric funnel plot may be caused by other reasons such as studies with low quality or small sample size (Fig. 6).

DISCUSSION
Our meta-analysis indicated a reduction of AAD from 16% in placebo to 13% in probiotic-treated groups (RR, 0.62; 95% CI, 0.51-0.74; random-effects). Further subgroup analyses suggested that the protective effect was still significant when grouped by reasons for antibiotics treatment, probiotic duration, probiotic dosage, and time from antibiotic to probiotic.
Compared with antibiotics treatments for other reasons, probiotics showed more effective protection during H. pylori treatment. Certain probiotics, when used as an auxiliary in H. pylori eradication, can increase the eradication rate and reduce side effects. 55 Meta-analyses for Saccharomyces boulardii and Lactobacillus both showed statistically promising results. S. boulardii significantly improved the eradication rates (RR, 1.11; 95% CI, 1.06-1.17) and reduced the incidence of diarrhea (RR, 0.51; 95% CI, 0.42-0.62). 56 So was the Lactobacillus (improving eradication rates: OR, 1.78; 95% CI, 1.21-2.62; reducing incidence of diarrhea: OR, 0.23; 95% CI, 0.11-0.48). 57 In terms of the mechanism of probiotics in H. pylori eradication, animal investigations have indicated that probiotics may regulate immune activity by controlling cytokine and inflammatory/ anti-inflammatory chemokine balance, such as interleukin-8 and secretory immunoglobulin A, thereby reducing gastric activity and inflammation. Also, probiotics assisted in promoting the H. pylori eradication through a physiological or nonspecific mechanism. Certain probiotics directly or in combination with their products stimulated gastric epithelium to produce antibacterial peptides, inhibited the growth of H. pylori by secreting short-chain fatty acids, competitively inhibited the adhesion of pathogens to the gastric mucosal layer, improved the epithelial barrier function, and increased mucin production. 58 We also explored the dose effect of probiotics in our meta-analysis. Our results showed that high-dose probiotics ( ≥ 10 10 CFU/d) were statistically less effective than lowdose probiotics (P = 0.05 < 0.10). However, a previous metaanalysis conducted by Johnston et al (involving adults and children) demonstrated that higher dosage ( > 10 10 CFU/d) had a more effective trend than lower dosage but not significantly (RR, 0.34; 95% CI, 0.23-0.49 vs. RR, 0.61; 95% CI, 0.08-4.60; P = 0.57 > 0.10). 59 This may be because we excluded children and the difference in sample size between subgroups. Hence, more RCTs on dose-response were needed to determine whether probiotics in higher doses were more effective and safe.
Our results are almost consistent with the previous meta-analysis in terms of the duration and starting time of probiotics. 60,61 It is beneficial to use probiotics as early as possible to maintain the gut flora's stability and prevent the overgrowth of pathogens. Concerning the optimal duration of probiotics, we suggested that probiotics use during antibiotic therapy can effectively prevent AAD. However, whether it is necessary to prolong the use of probiotics after the end of antibiotic treatment still needs more clinical evidence and theoretical support.
Twelve studies applied Lactobacillus as intervention indicated a more protective trend among all the probiotics species (RR, 0.67; 95% CI, 0.50-0.91). Among them,  62 This effect may be related to the colonization of LGG in the intestine. It not only enhances the survival rate of the intestinal epithelium survival and preserves cytoskeletal integrity, but also secretes lectin-like proteins 1 and 2 to resist biofilms produced by various pathogens. 63 Unfortunately, because of the insufficient sample size, some probiotics strains cannot be analyzed separately. In addition, we did not find significant differences in the efficacy of single species and multiple species (RR, 0.64; 95% CI, 0.44-0.93 vs. RR, 0.61; 95% CI, 0.49-0.76; P = 0.86 > 0.1). The type of antibiotic was reported as the strongest predictor for AAD. Although ampicillin/amoxicillin, cephalosporins, and clindamycin used alone were most frequently associated with AAD, other antibiotics, when used in combination, also increased the risk of AAD. 64 Unfortunately, many RCTs did not register specific antibiotics, which prevented us from performing subgroup analysis.
We extracted the data related to adverse events from 15 studies and thus calculated the pooled RR of 1.00 with no statistical significance (95% CI, 0.87-1.14; P = 0.97). A comprehensive systematic review on probiotics safety based on 622 studies displayed a pooled RR of 1.00 (95% CI, 0.93-1.07; P = 0.999), which was close to our finding. 65 These pieces of evidence were sufficient to show that short-term use of probiotics would not bring about serious side effects on a population without severe systemic disease or immunodeficiency. However, specific patients, including critical illness, using a central venous catheter, immunosuppression, should be sensitive to the adverse effects. 66 Some case reports and clinical studies have reported probiotics-related adverse events involving systemic infections, gastrointestinal side effects, deleterious metabolic activities, and gene transfer. 67 In short, probiotics are safe to use in preventing AAD.
There were some limitations. First, some heterogeneity was observed in our results. Both the subgroup analyses and sensitivity analysis failed to explain the source of heterogeneity. Second, some included studies failed to mention all specific characteristics. Thus, several subgroup analyses could not enroll all the 36 RCTs.
Nevertheless, our research also had some advantages. We adopted rigorous inclusion criteria to collect more representative data. During the citations identified, we excluded 2 publications with unknown probiotics composition. To avoid interference with baseline conditions, RCTs that included existing diarrhea or containing laxative-related diarrhea were also excluded. In addition, we conducted subgroup analyses as comprehensive as possible, and the trend of probiotics in some specific situations had been explored.
Our study suggests that using probiotics within 2 days during antibiotic treatment significantly reduces the incidence of AAD in adults and is safe. Besides, the existing evidence showed that S. boulardii supplementation or Lactobacillus supplementation in H. pylori eradication therapy significantly increased the eradication rate and reduced the incidence of diarrhea. But the role of other probiotics in H. pylori eradication had not yet been fully clarified. Of course, to match the population included in this meta-analysis, these findings are restricted to adults without immunodeficiency and the history of intensive care unit.

CONCLUSIONS
Our meta-analysis suggested that during antibiotic treatment, taking probiotics as early as possible has a positive and safe effect on preventing antibiotic-related diarrhea in adults. However, further studies should focus on the optimal dosage and duration of probiotics and pay attention to the strain specificity to develop a specific recommendation.