Acute diarrhea caused by pathogens may induce gastroenteritis, bloody stool, or severe intra-abdominal infections that establish disease and increase the economic burden, especially among infantile and childhood populations.
Probiotic Lactobacillus casei
can reduce the incidence of diarrhea and its duration in children [1
]. Moreover, the incidence was reduced among healthy young adults [3
] consuming yogurt containing Lactobacillus casei
. The mechanism by which probiotic bacteria, which inhabit the gastrointestinal tracts of healthy individuals, exert their beneficial effects is unclear. They may do so by enhancing host immunity, inhibiting bacterial epithelial and mucosal adherence, inhibiting epithelial invasion, and/or producing antimicrobial substances [4
Secretory IgA plays an important role in protection against infections caused by entero pathogens in both human and animal models [6
]. IgA is enhanced by probiotics as a reaction related to the mucosal immunity of the host intestine. Intestinal micro-organisms may play an important role in the development of the acquired immune system of infants, especially in the development of mucosal immunity and the production of endogenous IgA [7
Lactoferrin is a major whey protein, an 80 kDa iron-binding glycoprotein produced by many exocrine glands with a major constituent in the secondary granules of neutrophilic leukocytes. The presence of lactoferrin in body fluids, including intestinal lumen, is proportional to the flux of neutrophils and its assessment can provide a reliable biomarker for inflammation. Neutrophils have been shown to be involved in the perpetuation of inflammation in the gut in acute infection caused by bacterial pathogens as well as in inflammatory bowel disease (IBD) [8
]. Guerrant et al. confirmed increased fecal lactoferrin in 96% (25/26) of samples from patients with bacteria intestinal infection and concluded that fecal lactoferrin is a useful marker for fecal leukocytes [11
Calprotectin is a heterodimer of two calcium-binding proteins, S100A8 and S100A9. Both subunits are found at increased levels in the colon. Calprotectin is a 36 kDa calcium- and zinc-binding protein, and constitutes approximately 60% of soluble cytosolic proteins in neutrophilic granulocytes. Therefore, calprotectin is a marker of neutrophil influx and is elevated in a number of inflammatory conditions [12
]. Elevated calprotectin levels are a good predictor of inflammation, as well as being associated with standard inflammatory markers (e.g., serum C-reactive protein (CRP)). Fecal calprotectin is emerging as a useful marker to quantify mucosal inflammation, not in the least because it appears to be stable in feces, which can be obtained by noninvasive means [14
Our study sought to determine whether probiotics (Lactobacillus casei) inhibited gastrointestinal infection and reduced the associated inflammatory response. The bacterial counts and inflammatory parameters in fecal samples of acute childhood diarrhea were investigated. We assessed the effects of probiotics in the digestive system and fecal inflammatory markers of children with acute diarrhea.
2. Patients and Methods
2.1. Study Design
This prospective, randomized, case-controlled study analyzed children enrolled in Chang Gung Children’s Hospital located in Northern Taiwan. The study protocol was approved by the Institutional Review Board of Chang Gung Memorial Hospital, and the study was registered at ClinicalTrials.gov
(NCT 03856138). Informed consent was obtained from at least one parent or legal guardian prior to study inclusion. The study was performed in accordance with the Declaration of Helsinki. The study protocol was approved by the Institutional Review Board of Chang Gung Memorial Hospital, Taiwan. There are no conflicts of interest. Trial registration number: ClinicalTrials.gov
Subjects were divided into 2 groups (Lactobacillus casei variety rhamnosus treatment and control). Enrollment was carried out between December 2015 and February 2018. Diarrhea was defined as 3 or more outputs of loose or liquid stools per day. Inclusion criteria were aged 6 months to 6 years, and hospitalized children with suspicious infectious diarrhea of less than 72 h. Exclusion criteria were severe abdominal distension with risk of bowel perforation, immunodeficiency, risk for sepsis, underlying disorder with inflammatory bowel disease, history with surgical operation of the gastrointestinal tract, probiotic use in the 1 week preceding starting therapy, and antibiotics use in the hospital course.
Diet and probiotic micro-organisms: The preparation contained lyophilized powder Lactobacillus casei species. Each probiotic capsule contained 2 × 108 colony-forming units (CFU)/250mg of Lactobacillus casei variety rhamnosus (Lcr35®) in lyophilized powder form. The test capsule was commercially available (Probionov, Aurillac, France), and contained freeze drying bacteria (Lcr35®), the microbes of which were cultured in Man, Rogosa, Sharpe (MRS) broth, suspended in a lyophilization medium, subjected to freeze-drying, and then stored properly in a capsule. Each participant in the probiotic group consumed the lyophilized powder in capsule form twice daily (total cells: 4 × 108 CFUs/day) for 7 days over the course of the study. Both the probiotic and control groups were supplied by intravenous fluid hydration. Some clinical parameters were evaluated according to the following: body-weight change, appetite and daily intake, bloating or abdominal distension, abdominal or colic pain, diarrhea or fecal consistency, constipation, fever, and vomiting, which were also assessed. The appetite/intake score was evaluated by the parents or family of participants: 0, no oral intake; 1, little oral intake; 2, oral intake less than one-third of daily food; 3, oral intake more than one-third but less than two-thirds of daily food; 4, oral intake more than two-thirds of daily food; 5, very satisfied with oral intake.
2.3. Bacterial Culture and Probiotic-Bacteria Count
To assess the colonization of intestinal bacteria, fecal samples were collected from each patient on Days 0 (the day when patients were enrolled), 3, and 7, after probiotic or placebo treatment. The fecal specimens were weighed, homogenized, and serially diluted and plated on selective MacConkey agar for analysis of Gram-negative bacteria. After overnight incubation at 37 °C, bacteria colonies were counted. Bifidobacterium iodoacetate medium (BIM) agar plates were used to plate and analyze fecal Bifidobacterium counts. In order to assess the colonization effect of the Lactobacillus strain, fecal samples were also plated on deMan, Rogosa, and Sharpe agar (MRS; Difco), as well as Rogosa SL agar plates (Difco). In order to explore the total anaerobic bacteria counts, the fecal samples were also plated on anaerobic blood agar base plate (CDC-BAP) agar. After 48 to 72 h of anaerobic incubation at 37 °C, bacteria colonies could be confirmed with regard to colonization. Fecal bacteria count was expressed as log10 CFU/g feces.
2.4. Taxonomy-Based and Statistical Analyses
Approximately 0.2 g of a wet-sludge sample pellet was used for DNA extraction with a QIAamp® Fast DNA Stool Mini Kit (Hilden, Germany), according to the manufacturer’s instructions. The extracted genomic DNA samples were examined by 1.5% agarose gel electrophoresis. Then, they were stored at −20 °C until use.
2.4.1. 16 S rRNA Amplification
For each fecal sample, a 50 μL polymerase chain reaction (PCR) mix was prepared containing 25 ng DNA template, 5X KAPA HiFi Buffer, 10 mM KAPA dNTPMix, 1 U/μL KAPA HiFi DNA Polymerase (KAPA Biosystem, Boston, United States), and 0.3 μM of each primer (Tri-I, New Taipei, Taiwan). PCR reaction conditions consisted of an initial 95 °C for 3 min followed by 15–25 cycles of 98 °C for 20 sec, 45 °C for 15 sec, and 72 °C for 15 sec, and a final extension of 72 °C for 1 min. The PCR products were purified with a QIAquick PCR purification kit (Qiagen, Hilden, Germany), quantified using a Qubit® dsDNA HS assay kit (Invitrogen, Grand Island, NY, USA) on a Qubit® 2.0 Fluorometer (Invitrogen, Grand Island, NY, USA). PCR products were sequenced on an illumina sequencing platform according to the instructions of recommended Miseq procedures.
2.4.2. Library Construction and Illumina MiSeq Sequencing
The paired-end library was constructed with Ovation® Ultralow DR Multiplex System 1–96 (NuGen, San Carlos, CA, USA), all according to the respective manufacturers’ instructions. Library concentration and quality were assessed on a Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA) using a DNA 1000 lab chip (Agilent, Palo Alto, CA, USA). Then, 16S amplicon libraries were sequenced 2 × 301 + 8 bp (index) by using a Miseq Reagent kit v3 (600 cycles) on an Illumina MiSeq system following the manufacturer’s instructions.
2.4.3. Data-Processing Pipeline
The sequencing reads were initially demultiplexed using a MiSeq Reporter v2.6 according to the sample barcodes. The resulting pairs of reads from each sample were merged to obtain longer reads (460 ± 50 bp) to improve taxonomy classification using FLASH (V1.2.11) [15
]. Only samples with merged reads ≥100,000 were retained for subsequent analysis. Low-quality reads with q-value <20 were filtered by the split_libraries_fastq.py script of QIIME (version 1.9.1) [16
]. Only sequence tags with length >400 bp were retained for subsequent analysis. Operational taxonomic units (OTUs) were clustered at 97% sequence similarity using USEARCH (v9.2.64) [17
] against the Greengenes 16S rRNA database (13_8 release), and the final taxonomic assignment was performed using an RDP classifier [18
]. Clustal Omega software was used to construct phylogenetic trees from representative OUT sequences.
2.5. Fecal Immunoglobulin Assay
Fecal samples were collected during the treatment period. IgA levels were measured on homogenized fecal samples. Immuno II plates were coated with goat antihuman IgA (1μg/mL) and incubated for 60 min at room temperature. After blocking and washing, the plates were incubated with diluted fecal supernatant samples and standard human reference serum IgA Calibrator (Bethyl Laboratories, Inc). Total IgA was determined using a goat antihuman IgA-HRP conjugate. The reaction was developed with tetramethylbenzidine (TMB; Zymed Labs.) and read at 450 nm. OD values were converted to ng/g feces of total IgA by comparison with a standard curve developed with antihuman IgA.
2.6. Measurement of Inflammatory Mediators in the Intestine
Fecal samples of these patients were collected at 3 different time points, including the initial stage of acute diarrhea (the day when patients enrolled), 3 days later, and 7 days later. Series follow-up of fecal lactoferrin and calprotectin were measured by ELISA. Fecal extracts were prepared by mixing stool (1 g of feces in 4 mL of buffer) with phosphate-buffered saline (PBS) containing ethylenediaminetetraacetic acid (EDTA, 0.05 M), the protease inhibitor soybean trypsin (100 μg/mL), and phenylmethylsufonyl fluoride (10 mM). Fecal extracts were frozen in aliquots at −70 °C.
Lactoferrin assay: Stool samples were prepared and analyzed for lactoferrin according to the manufacturer’s instructions (AssayMax Human Lactoferrin ELISA Kit, St. Charles, MO, USA). This assay employs a quantitative sandwich enzyme immunoassay technique that measures lactoferrin in 4 h. A polyclonal antibody specific for lactoferrin was precoated onto a microplate. Lactoferrin in standards and samples was sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for lactoferrin, which is recognized by a streptavidin–peroxidase conjugate. Absorbance was read at OD 450 nm. Lactoferrin was expressed as μg/g feces.
Calprotectin assay: Stool samples were prepared and analyzed for calprotectin according to the manufacturer’s instructions (PhiCal
Calprotectin ELISA Kit, Immundiagnostik, Bensheim, Germany). Stool was collected in plastic containers and sent to the laboratory by mail either the same or the next day. The weight of each sample (100–500 mg) was measured, and an extraction buffer containing citrate was added in a weight/volume ratio of 1:50. The samples were mixed by vortex mixer for 30 s and homogenized for 25 min. One milliliter of the homogenate was transferred to a tube and centrifuged for 20 min at 10,000× g
. Finally, the supernatant was collected and frozen at –20 °C.The supernatants were thawed, and calprotectin was analyzed with the quantitative calprotectin ELISA and read at OD 450 nm. Calprotectin was expressed as μg/g feces [19
2.7. Statistical Analysis
Comparison between groups was performed with the t-test or the Mann–Whitney U test, the [chi]2 test, and ANOVA for repeated measures. Analyses were performed on the intention to treat the population. To explore time, treatment, and their interaction, a 2-factor ANOVA for repeated measures containing 3 time points and 2 groups, and the Tukey–Kramer post hoc test were used to analyze the data after verifying the normal distribution of the dataset. A significance level of 0.05 was used, and the statistical tests were two-tailed. GraphPad Software Prism 3.03 (GraphPad Software, Inc., San Diego, CA, USA) and SPSS Software, version 18.0 (SPSS Inc., Chicago, IL, USA), were used for statistical analysis.
Acute diarrhea is a major cause of childhood morbidity, with an emotional and economic burden for the families of affected children, as well as a burden on society [20
]. Several types of medications, such as those affecting intestinal motility, ion transport, and adsorptive moieties, as well as living bacteria have been used in an attempt to reduce the duration of acute diarrhea [21
]. Our study assessed the clinical effectiveness and fecal parameters of a commercially available probiotic product.
In our study, probiotics can reduce the severity of diarrhea with statistical significance (p
< 0.05). Our findings were consistent with previous studies showing probiotics to be effective in reducing the duration of diarrhea [22
]. Moreover, bowel habits improved under probiotic colonization. Probiotics also reduced abdominal pain or colic accompanying diarrhea episodes after the start of the treatment, as well as bloating or abdominal distension, especially on the day 7 and day 14 follow-ups. In our study, parents of children that were given probiotics seemed more satisfied with the better appetite and oral intake on the daily record, and even better weight gain.
However, some studies [26
] found no positive effect of Lactobacillus casei
on the clinical course of diarrhea, such as antibiotic-associated diarrhea. The possible reasons of why probiotics failed to do so are that the dosage, species of probiotics, or antibiotics therapy may reduce the anaerobic bacteria of the intestinal flora. In a recent report [28
], compared to spontaneous post antibiotic recovery, a probiotic mixture containing at least 2.5 × 1010
active bacteria induced a delayed and persistently incomplete indigenous stool/mucosal microbiome reconstitution. Probiotics may also negatively influence the recolonization of commensal bacteria in antibiotics given to subjects [28
Prominent differences were also observed in the diversity and composition of the gut microbiota of responders versus non-responders in immunotherapy for melanoma [30
]. Moreover, some challenges remain to be solved concerning how to regulate gut microbiota to enhance the efficacy of cancer immunotherapy. Which specific composition of the gut microbiota is most conducive to ameliorating an anti-tumor immune response is unclear, and there are a variety of management options that alter gut microbiota [32
]. The use of probiotics and antibiotics requires careful consideration. These long-term limitations of probiotics use should be addressed in specific situations, such as immunodeficiency individuals, patients undergoing anti-tumor therapy, or patients receiving a long course of antibiotics therapy.
Children usually suffer pain when collecting a blood sample through venipuncture or investigating with invasive endoscopy techniques. Noninvasive tests such as those involving stool samples are highly recommended for children and can help avoid pain. We; therefore, collected fecal samples and performed further analysis of bacteria counts and other inflammatory mediators. Previous studies showed that supplementation with Lactobacillus
] restores the total Lactobacillus
counts in feces, respectively. In our study, probiotic preparations (Lactobacillus casei
) may promote the major population of Lactobacillus
in stool output, as well as Bifidobacterium
counts in feces.
is a Gram-positive anaerobic gut microbial genus in the Clostridia class and Ruminococcaceae family. Lachnospiraceae and Ruminococcaceae are two of the most abundant families from the Clostridiales order found in the mammalian gut environment, and they have been associated with the maintenance of intestinal health [36
]. Lachnospiraceae such as Eubacterium rectale
, Eubacterium ventriosum
spp., and Roseburia
spp. have been associated with the production of butyrate, which is the major energy source of colonic epithelial tissue and may be involved in immune regulation and intestinal-barrier function [37
]. In contrast, recent studies [41
] have shown that butyrate is not universally beneficial for intestinal health. Lachnospiraceae and Ruminococcaceae differ with respect to their average numbers of carbohydrate-active genes and gene families, particularly glycoside hydrolases and carbohydrate-binding modules, which are more abundant and more diverse in Lachnospiraceae and Ruminococcaceae [43
]. Our results revealed a higher abundance of Ruminococcaceae and Lachnospiraceae in children with the probiotic supplement.
Primary fermenters, such as Bacteroides
, are gateways through which carbohydrates enter the network of metabolic interactions. Bacteroides
degrade complex carbohydrates to their component monosaccharides, which, in turn, are metabolized through the sequential actions of metabolic pathways. The main excreted products of Bacteroides
are acetate, propionate, and hydrogen [44
]. Acetate, propionate, and butyrate are end-products of microbial action on endogenous and exogenous dietary and host-derived components. The protective effect of commensal bacteria may be regulated by the above metabolic products. Our results revealed a higher abundance of Bacteroides
in children with the probiotic supplement.
Enterobacteriaceae are a prominent family of Gram-negative bacteria, which belong to the Enterobacteriales order and the Proteobacteria phylum. Enterobacteriaceae include many familiar pathogens, such as Shigella, Salmonella
, Escherichia coli
, and Yersinia
. Other disease-causing bacteria in this family include Proteus, Serratia, Enterobacter
, and Citrobacter
]. Our results revealed a lower relative abundance of Enterobacteriaceae in children with the probiotic supplement.
The function of fecal IgA is to agglutinate micro-organisms and to prevent the adherence of pathogenic microorganisms to the mucosal surface, as well as maintenance of intestinal microbial homeostasis [49
]. There is a close interaction between the composition of intestinal micro-organisms and IgA production. Simultaneously, the continuous presence of commensal bacteria (e.g., probiotics) favors a constant stimulation of the immune system without invoking inflammatory responses [52
]. In our study, total IgA levels in the fecal extracts of the Lactobacillus
group were higher (p
< 0.05) on Days 3 and 7 after therapy initiation than those in the control group. This result is a remarkable finding in probiotics treating acute childhood diarrhea, indicating that probiotics may enhance the protective immune response in the intestinal tract of the host.
Cells of the innate immune system secrete various enzymes and metabolites, including myeloperoxidase and lactoferrin, produced by activated neutrophils. Lactoferrin is mainly found in the oral cavity and intestinal tract where it can come into direct contact with pathogens such as viruses and bacteria. The most significant function of lactoferrin in mucosal defense is its antimicrobial activity. Lactoferrin can also amplify the actions of lysozyme and secretory immunoglobulin A [53
]. Noninvasive fecal lactoferrin may prove useful in screening for inflammatory intestine activity in patients with abdominal pain and diarrhea [54
]. According to our study, fecal lactoferrin levels were lower in patients with the probiotic treatment, indicating that probiotics attenuate inflammatory activity in the intestinal tract of the host.
A remarkable intestinal infection is the intense infiltration of neutrophils, macrophages, lymphocytes, and other inflammatory cells in the epithelial lining and the lamina propria of the colonic mucosa [56
]. Neutrophils have been shown to be involved in the perpetuation of gut inflammation in acute infections caused by bacteria [8
]. Fecal calprotectin, in particular, has for a long time been regarded as a promising marker of gastrointestinal pathology [57
]. Fecal calprotectin has been proposed as a useful marker of gastrointestinal inflammation [19
]. There are some studies that have explored the role of fecal calprotectin in acute infectious diarrhea. One study [58
] enrolled children of less than five years of age. The concentration of fecal calprotectin correlates with intestinal inflammation, and children with bacterial gastroenteritis were found to have higher fecal calprotectin compared to viral gastroenteritis. Another study [59
] investigated adult diarrhea and showed higher levels of calprotectin in bacterial infections. In our study, fecal calprotectin levels were lower in patients with probiotic treatment compared to those in the control. The above finding suggests that probiotics downregulate inflammatory activity in the intestinal tract of the host.