The Role of Oral Probiotics in Alleviating Inflammation, Symptom Relief, and Postoperative Recurrence and Their Side Effects in Adults With Crohn's Disease: A Systematic Review

Crohn's disease (CD) is a lifelong problem for patients, despite having multiple pharmacological options and surgeries for treatment. In order to achieve best results, probiotics are being used even though their efficacy is still debatable. This systematic review analyzes the safety and efficacy of several probiotics in CD. PubMed, the Cochrane Library, and ScienceDirect are the databases searched for randomized controlled trials (RCTs), animal studies, in vitro studies, and reviews. After quality appraisal and cross checking the literature, this systematic review is carried out grounded on Preferred Reporting Items for Systematic Review and Meta-Analysis 2020 (PRISMA 2020) guidelines. A study of 16 papers in total which include nearly 2023 subjects showed that only very few probiotics are efficient in furnishing remission in CD complaints. Kefir, an inexpensive fermented milk product, significantly reduced the inflammation and drastically bettered the quality of life and hence can be considered as an asset for CD patients. Lactobacillus thermophilus, Bifidobacterium longum, Enterococcus faecalis, and Bacillus licheniformis can control diarrhea in patients of 22-54-year age group and improve cognitive reactivity in sad mood with short-term consumption. VSL#3 (VSL Pharmaceuticals, Gaithersburg, Maryland, United States) has good efficacy in precluding recurrence and easing side effects after ileocecal resection in adults. Animal models and lab studies have proved that Lactobacillus plantarum CBT LP3, Saccharomyces cerevisiae CNCM I-3856 (yeast), few strains of Lactobacillus plantarum, Bifidobacterium animalis spp., Lactobacillus acidophilus LA1, Lactobacillus paracasei 101/37, and especially Bifidobacterium breve Bbr8 are significant enough to ameliorate the disease condition. In conclusion, probiotics are safe in CD with very few modifiable side effects. Some probiotics are proven to be significant in animal and lab studies; hence, these should be studied in human RCTs, to check their efficiency in human beings. There are limited observational and interventional studies in this regard. Large population-sizes trials are highly demanded in the areas of prognosticated positive results that are mentioned in this systematic review.


Introduction And Background
Inflammatory bowel disease (IBD) that includes Crohn's disease (CD) and ulcerative colitis (UC) is a complaint of lifetime and deteriorates the quality of life of patients and their families substantially [1].1.3% of US adults, which is three million, were reported to be diagnosed with IBD in 2015 [2].The prevalence in the United States is estimated at 58 per 100,000 children and 119-241 per 100,000 grown-ups, and it is increasing in both age groups.Although new cases occur in after periods, most cases are diagnosed in the twenties and forties.Prevalence is more in the white race.6.3 billion dollars is the estimated annual economic burden to the US healthcare system [3][4][5][6].In China from 1994 to 2004, the incidence had increased by threefold [7].According to the World Journal of Gastroenterology, areas with the highest incidence are the United Kingdom, North America, and the northern part of Europe [8,9].The prevalence of CD in Europe varies from <10 to about 150 per 100000 occupants [10,11].
In 2017, there were 6.8 million cases of IBD globally, and the age-formalized death rate is 0.51 (0.42-0.54) per 100,000 population.Loftiest age-standardized prevalence rate was seen in high socio-demographic index (SDI) locations.From 1990 to 2017, the prevalence of IBD increased substantially in many regions which might pose a significant social and financial burden on governments and health systems in the coming times [12].CD, a chronic IBD, can affect any portion of the gastrointestinal tract.It manifests as spastic granulomatous patches due to effects of environmental factors, inheritable predisposition, and mucosal immunity.Common presenting symptoms are abdominal pain, rectal bleeding, weight loss, fever, fatigue, and diarrhea.
Uncontrolled inflammation may lead to long-term complications such as fibrotic strictures, enteric fistulae, and intestinal neoplasia.Therefore, early and effective control of inflammation is of paramount value [13,14].
The etiology of CD is multifactorial; thus, understanding only one aspect of IBD pathogenesis does not reflect the complex nature of IBD, nor will it enhance its clinical management.Therefore, it is pivotal to segregate the interactions between the various factors in IBD pathogenesis [15].Several studies have reported microbial dysbiosis and immune and metabolic dysregulation in IBD cases; however, this data is not sufficient to produce signatures that can differentiate between complaint relapse and remission.The current knowledge gap in IBD hinders the advancement of clinical decision for treatment, as well as the prediction of disease relapse.There has been exploration going on regarding the use of prebiotics, probiotics, and synbiotics independently and in combination with other drugs like 6-mercaptopurine (6MP), aminosalicylates, corticosteroids, antibiotics, and surgical procedures to ameliorate the condition [15].
The management of CD and UC has changed drastically over the last two decades due to the introduction of targeted biological therapies, but the impact of these new medicines in altering the natural history of disease is still under debate.Latest evidence seems to suggest that their efficacy might be incompletely dependent on the timing of their introduction and further management of the disease.So far, the potential role for a more dynamic approach with treatments grounded on sequencing and combining targeted preventive and curative therapies has been explored only to a minimal extent.
Despite the available treatment strategies, a significant number of cases suffer one or multiple surgeries in their continuance.The current pharmacological treatment has variable efficacy and is associated with the threat of significant side effect; hence, there is a constant need to search for new types of therapies with optimum safety standard [16].The arrival of biologics has clearly represented a good advance in IBD treatment, but their efficacy in changing the natural history of the disease has not been clearly established [8].In view of the above factors, we aim to explore in this systematic review the efficacy of probiotics in remission, inflammation, symptom relief, and postoperative recurrence and their side effects in adults with CD based on the recent research.More importantly, we are fastening on the stylish probiotic preparations available with considerable evidence from the latest works, to be recommended for the adult patients in agony suffering from CD.

Review Methods
This manuscript is written according to the Preferred Reporting Items for Systematic Review and Meta-Analysis 2020 (PRISMA 2020) guidelines [17].Table 1 shows the total number of articles available on different databases regarding the role of probiotics in CD.

Inclusion and Exclusion Criteria
Studies comprising pediatric age group patients, pregnant women, and critical cases are removed.Studies on IBD with less focus on CD are removed as well.Probiotics given by other means like fecal transplantation and cancer-related papers are excluded also.

Quality Appraisal
The Cochrane bias assessment tool for randomized controlled trials (RCTs), A MeaSurement Tool to Assess systematic Reviews (AMSTAR) checklist for systematic reviews, the SYstematic Review Centre for Laboratory animal Experimentation (SYRCLE) for animal studies, and the Scale for the Assessment of Narrative Review Articles (SANRA) checklist for other studies are the tools used for quality check.The list of selected studies with the year of publication, study type, and number of subjects involved is presented in Table 2.The effects of various probiotics on CD are assessed in this systematic review which include disease remission and symptom relief, improving the quality of life of the patients, effect on Crohn's Disease Activity Index (CDAI), effects on inflammation, emotional symptoms, bowel functions, systemic functions, wellbeing, abdominal pain, abdominal mass, quantity of liquid stools, hemoglobin levels, hematocrit, white blood cell (WBC) counts, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), calprotectin levels, impact on diarrhea thus on mood disorders, efficacy in altering microflora in colitis, postoperative recurrence, disease relapse in steroid or aminosalicylate users, histopathology, immunomodulation, and side effects.The review of various RCTs, lab and animal studies, and review studies are analyzed in Table 3, Table 4, and Table 5, respectively, that include the sub-topics discussed, types of probiotics used, other drugs given for the studies, and their results along with their side effects.

Placebo
Statistics showed no significant modulation of microbiome between cases and controls, the results are not only insignificant but also erratic, and the median age of patients is 62 years.No side effects are discussed; however, the population size is small and old aged, and there is no washout period between switching the groups.At day 90 (after phase 2), the proportion of patients with endoscopic recurrence is 9.3% in VSL#3-treated group and 15.3% in the placebo group with p=0.19, and the rate of recurrence is also not significant (p=0.82).At day 365 (after phase 3), there was no severe recurrence in 89.6% of previous VSL#3 users continuing VSL#3 in phase 3, and there is no severe recurrence in 71.4% patients from the placebo group who are taking VSL#3 in phase 3 but did not take VSL#3 in phase 2. Severe recurrence rate in early VSL#3 group is 20.5% and in early placebo group is 42.1%.Colonic mucosal cytokine release is also less in patients who started VSL#3 immediately and high in immediate placebo groups (took VSL#3 lately).Side effects: In the early placebo + late VSL#3 group, two patients had small bowel obstruction due to adhesion, one patient faced worsening of disease, one patient experienced postoperative wound infection, there was one case of ventral hernia repair, and there was one case of traumatic stabbing, whereas only one case of post-op wound infection is seen in the early + late VSL#3 group.In the experiments, the effects of AIEC LF82 invasion of IECs and CCR6-CCL20 axis were tested.Again, to verify that LF82 inhibition is not simply due to any bacterial strain, co-infection experiments with S.
epidermidis ATCC155 and also with 6MP were conducted, and the results showed that L1, L2, B1, and B2 significantly reduced AIEC LF82 adhesion to human colorectal adenocarcinoma cell line with epithelial morphology (HT29 cells) by blocking specific adhesion determinants and enhanced biofilm formation.Although Bifidobacterium, E. coli, and Lactobacillus were used for maintaining therapy in different trials, they did not show any favorable effect over controls in patients with CD.
Mucosa-associated microbiota in CD patients exhibited reduced bacterial richness and diversity, while there was a clustering of patients and healthy controls.Machiels established that fecal and mucosal microbiota constitute different ecological environments before surgery.No bacterial taxon altered counts consistently.The alterations in mucosal microbiota are more significant in Bacteroidetes and Firmicutes phyla, but less significant in Proteobacteria phylum; also, the expansion of Fusobacteria, a putative aggressive phylum, was also observed.The factors influencing bacterial populations following ileocolonic resection are substantial catabolic stress, retrograde flow of colonic contents, inflammatory changes involved in intestinal wound healing, and altered immune function.At the time of postoperative endoscopy, the population of Actinobacteria, a proteolytic bacterial phylum with the capacity to invade and exacerbate inflammation, was depleted in patients with CD; however, the phylum Fusobacteria maintained its higher relative abundance, while the alterations in the phylum Bacteroidetes count were inconsistent.
Higher abundance of Enterococcaceae and Fusobacterium and lower abundance of Lachnospiraceae and Faecalibacterium existed in both postoperative mucosal and fecal microbiota in CD patients.As fecaland mucosal-associated microbiota constitute different ecological environments, they have different

Veillonella
Antibiotics and corticosteroids alteration trends.Notably, the presence of mucosal bacterial genera, such as Bacteroides, Prevotella, and Parabacteroides, which are associated with saccharolytic metabolism, has been correlated with increased remission compared to the presence of bacterial genera, such as Enterococcus and Veillonella, which are associated with fermentation and lactic acid production.However, no specific bacterial taxa are consistently different between CD patients with or without PR in any of the included studies.Several commensal bacteria with lower relative abundance are known to exert an anti-inflammatory effect by decreasing colonic pro-inflammatory cytokine synthesis and inducing anti-inflammatory cytokine secretion.
In addition, patients with recurrent CD retain microbiota that favors proteolytic-fueled fermentation and lactic acid production, while CD patients in remission retain a predominantly saccharolytic and SCFAproducing microbiota.Furthermore, changes in the ecology of depleted SCFA-producing bacteria may permit the expansion of pathogenic bacteria through luminal environmental perturbation (deviation from normal).Till date, no study has evaluated the role of the metabolomic profiling of gut microbiota at the time of surgery or at the postoperative follow-up in the identification of metabolites that may be associated with CD recurrence after intestinal resection.The number of published studies investigating the involvement of gut microbiota both in monitoring the postoperative disease progression and in assessing the response of CD patients to a treatment is surprisingly low.There are only four studies that provide information on the potential of gut microbiota to predict the PR of CD, but their results were too heterogeneous to make a confident conclusion regarding a microbial biomarker.High abundance of bacteria from the Proteobacteria phylum (e.g., Proteus and Ralstonia) as well as in Ruminiclostridium gnavus (Gammaproteobacteria) and Corynebacterium and the reduced abundance of several members of the Firmicutes phylum, particularly the Lachnospiraceae and the Ruminococcaceae families (e.g., Faecalibacterium, Gemella, Phascolarctobacterium, Coprobacillus, unidentified Lachnospiraceae, and Dorea), were predictive of endoscopic PR.Keshteli's study says distinctive urinary metabolomic profiling associated with Bacteroidales and Gammaproteobacteria has the potential to be used as a biomarker for the identification of CD patients who develop endoscopic disease recurrence after ileocolonic resection.Study by Campieri reported that the combination of a non-absorbable antibiotic (rifaximin) and a highly bacterial concentrated probiotic (VSL#3) is efficient in preventing the severe endoscopic recurrence of CD.Early postoperative use of antibiotics to prevent pathogenic recolonization followed by maintenance with the use of probiotics to establish a durable anti-inflammatory postoperative microflora may yield the greatest benefit with the least risk of disease recurrence in CD patients.
Unfortunately, the efficacy of fecal microbiota transplants in preventing the PR of CD remains unknown.
Exclusive probiotic therapy is not found to be effective in preventing PR.Antibiotics are superior over probiotics in this regard.Insufficient trials.

Discussion
Almost 2023 cases are studied from all the 16 papers, and only very few probiotic preparations are found worth of using in adult CD patients based on the available latest evidences.There are certain probiotics that showed mind-relaxing results.
Yılmaz et al. [19] with 45 subjects in the year 2019 established that consumption of kefir, a fermented milk product, in adults showed excellent symptom relief which is statistically significant.It also improved quality of life and reduced inflammation.Moreover, its consumption had no lactose intolerance-related effects; hence, kefir is safe, affordable, and very effective with short-term consumption which proves it to be an asset for CD patients.We explosively recommend further large population-sized clinical trials with kefir as we anticipate the same results.The effect of kefir in long-term consumption has to be studied.
On the other hand, Bourreille et al. [23] in the year 2013 studied 125 cases and proved that Saccharomyces boulardii had no side effects.Location of the disease in the gastrointestinal tract of the patients has no influence on the risk of relapse.However, this study excluded severe CD patients, and mucosal healing is not studied systematically.Biological parameters were used to assess inflammation.S. boulardii at a daily dose of 1 gram, although safe and well tolerated, did not show any beneficial effect as a preventive remedy in patients with moderate and severe CD.No effect on dwindling side effects was noted.
Bjarnason et al. [18] in the year 2019 with 250 participants showed that Symprove (an admixture of Lactobacillus rhamnosus NCIMB 30174, Lactobacillus plantarum NCIMB 30173, Lactobacillus acidophilus NCIMB 30175, and Enterococcus faecium NCIMB 30176) taken for four weeks in asymptomatic CD patients is not effective in symptom relief and improving quality of life and perfecting emotional symptoms, but minimally effective in preventing re-occurrence.Wu et al. [20] in the year 2021 with 46 subjects reported that Lactobacillus thermophilus, Bifidobacterium longum, Enterococcus faecalis, and Bacillus licheniformis are safe and have strongly controlled the diarrhea symptoms through regulating the gut microbiota in the CD patients between 22 and 54 years of age and also had better cognitive reactivity to sad mood in three weeks.
Ahmed et al. [21] in the year 2013 with very small population size demonstrated that Trevis capsule that contains four strains of probiotics, Lactobacillus acidophilus LA-5, Lactobacillus delbrueckii subsp.bulgaricus LBY-27, and Bifidobacterium subspecies is safe, but had no beneficial effects on old-aged people in CD in altering colonic microflora.Fedorak et al. [22] in the year 2014 with 119 subjects showed that commencing VSL#3 soon after surgery might not show better results after few months, but prevents recurrence with consumption for one year and crucial in easing numerous side effects in 16-yearold or above aged patients who underwent ileocolonic resection with a small-intestine-to-colon anastomosis.VSL#3 is safe in CD.Kim et al. [24] in the year 2020 demonstrated that, in colitis-induced mice, L. plantarum CBT LP3 is largely effective in alleviating disease activity, weight loss, and inflammation.Histopathological damage is very much reduced.It is effective in preventing relapse as well.Although the population size is small, based on the results of this study, it is recommended to conduct analogous experiments in animals with bigger population size, and then with at most safety precautions, human trials could be conducted to demonstrate the use of L. plantarum CBT LP3 in CD colitis.Side effects are to be studied in human trials.
In 2015, Sivignon et al. [25]  In vitro study by Leccese et al. [27] in the year 2020 suggests that the efficacy and safety of Bifidobacterium animalis spp.Lactis Bi1 (B1), Bifidobacterium breve Bbr8 (B2), Lactobacillus acidophilus LA1 (L1), and Lactobacillus paracasei 101/37 (L2) (especially B2) should be studied in AIEC LF82-induced CD by conducting RCTs as they significantly controlled the adhesion of AIEC LF82 and enhance biofilm formation.Chen et al. [28] in the year 2021 reviewed papers with 289 CD patients and showed that VSL#3 that contains Lactobacillus strains (L.paracasei DSM24733, L. plantarum DSM24730, L. acidophilus DSM24735, and L. delbrueckii subsp.bulgaricus DSM24734), three strains of Bifidobacterium (B.longum DSM24736, B. breve DSM24732, and B. infantis DSM24737), and one strain of Streptococcus salivarius subsp.thermophilus DSM24731 showed no significant results.The results showed that S. boulardii, L. johnsonii LA1, and L. rhamnosus GG have no remarkable goods in the remission and recurrence of CD.Study by Dore et al. [29] in the year 2019 conducted a systematic review that involved 178 patients to estimate the side effects of Lactobacillus rhamnosus GG, L. johnsonii LA1, B. longum, and Synergy and showed that, if the unpleasant taste is changed using required techniques, oral probiotics are more comfortable and much safe in CD.Lorentz and Müller [30] in the year 2022 conducted a systematic review with 482 subjects involved, out of which 32 were CD cases.This review established that S. boulardii reduces recurrence in mesalazine users in CD.Limketkai et al. [31] in the year 2020 conducted a systematic review that involved 45 subjects and showed that Bifidobacterium longum and Synergy are ineffective in CD remission.
A meta-analysis conducted by Shen et al. [32] in the year 2023 which involved 43 subjects, studied effects of probiotics on the remission of CD, and showed that Bifidobacteria, E. coli, and Lactobacillus were used for maintaining therapy in different trials did not show any favorable effect over controls in patients with CD.
No significant effect on problem remission was seen.Zhuang et al. [33] in the year 2020 conducted a systematic review with 809 patients to check the role of several probiotics in postoperative recurrence, but no significant results were retrieved.Exclusive probiotic therapy is not found to be effective in preventing postoperative recurrence.Antibiotics are found to be superior to probiotics in this regard.Trials are regarded to be inadequate.

Conclusions
This systematic review studies the safety and efficacy of various probiotics in CD remission and postoperative recurrence and their side effects from the latest available research from several databases.We excluded CD patients that developed cancer, pregnant women, and ICU patients.The latest available quality data from the last 10 years shows that kefir which is a fermented dairy product is an asset for adult CD patients.L. thermophilus, B. longum, E. faecalis, and B. licheniformis are safe and have strongly controlled the diarrhea in patients between 22 and 54 years age and also have better cognitive reactivity to sad mood in three weeks.VSL#3 prevents recurrence and side effects in adults undergoing ileocecal resection.These results in turn must be tested in large population-sized trials in order to establish their effects on a large scale.Few probiotics proven very effective in animal and in vitro studies are to be tested in human trials, and there is a big scope based on these results that testing them in human trials is worth sought after.S. boulardii reduces recurrence in mesalazine users in CD.The unpleasant taste of oral probiotics could be changed to increase their use.Hence, we reckon the presentation of evidences and interpretation of effects are precise enough albeit there are less number of clinical trials and very less observational studies available regarding the role of probiotics in CD in human beings.

TABLE 2 : List of studies and their characteristics
RCT: randomized controlled trial; CD: Crohn's disease

TABLE 3 :
RCTs At day 7, the survival rate of LF82-infected mice is 30%, and yeast-treated mice is up to 70%.Histological scores: Untreated is 5.6+/-0.9,treated is 2.4+/-0.5, and p-value is <0.01 (nearly same as normal mice).Massive neutrophilic infiltration with transmural involvement, lymphoepithelial lesions, and mucosal erosions are seen in the non-treated group, but not seen in the treated group.MPO activity is altered but not significant.Intestinal permeability restored to almost basal levels.The overexpression of CLAUDIN-2 (a pore-forming tight junction protein) associated with plasma membrane is resolved.Effects of treating infected mice with cell wall derivatives of yeasts rather than live yeasts: Less severe BW loss seen (direct yeast is 90.2% and with derivatives is 83.9%) and DAIS is not significant after days 3 and 4.

TABLE 4 : Animal and in vitro studies
Placebo CD recurrence reviewed from four studies that include 289 subjects (145 probiotic users and 144 other drug users) showed statistically insignificant results with a p-value of 0.52.In remission, the probiotics showed some effects, and no side effects were mentioned.No side effects are mentioned where 12 billion cfu/day probiotics was given for 52 weeks.Study 2: 2x10 9 cfu/day for six months was given.Abdominal pain and unpleasant taste were reported.Study 3: 2x10 11 cfu/twice daily with 6 grams of fructooligosaccharides or inulin was given for six months.Out of 35 patients, only two patients could not tolerate synbiotics.
[26]ied the efficacy of Saccharomyces cerevisiae CNCM I-3856 (yeast) in preventing colitis induced by adherent invasive Escherichia coli (AIEC) LF82 in mice, and very significant results were obtained; pre-incubation results are more prominent than co-incubation and post-incubation effects.Such appreciable results are not seen with the cell wall components of AIEC LF82.Significant results are observed in reducing disease activity index and quality of life.Human trials are needed to study the side effects and further confirmation of findings: if S. cerevisiae yeasts could prevent CD in patients abnormally expressing CEACAM6 at the ileal mucosa.Study by Choi et al.[26]in 2019 concluded that in lipopolysaccharide-induced RAW264.7 cells and a dextran sulfate sodium (DSS)-induced colitis animal model, the effects of probiotics are as follows: Lactobacillus plantarum CAU1064, CAU1031, CAU1273, and CAU1054 strains have nitric oxide (NO) and cyclooxygenase-2 (COX-2) suppression potential.