Compliance to gluten-free diet may regenerate probiotic microbiota: First report

Moni Kumari; Malika Arora; Navdeep Kaur; Ranjit Singh; Parveen Bansal

doi:10.4103/jihs.jihs_34_20

ORIGINAL ARTICLE

Year : 2021 | Volume : 9 | Issue : 1 | Page : 8-12

Compliance to gluten-free diet may regenerate probiotic microbiota: First report

Moni Kumari¹, Malika Arora², Navdeep Kaur³, Ranjit Singh⁴, Parveen Bansal⁵
¹ Multidisciplinary Research Unit, Guru Gobind Singh Medical College, Faridkot and Adarsh Vijendra Institute of Pharmaceutical Sciences, Saharanpur, Uttar Pradesh, India
² Research Scientist II, Multidisciplinary Research Unit, Guru Gobind Singh Medical College, Faridkot, Punjab, India
³ Department of Microbiology and Biotechnology, School of Biological Engineering and Life Sciences, Shobhit Institute of Engineering and Technology, Meerut, Uttar Pradesh, India
⁴ Director, Adarsh Vijendra Institute of Pharmaceutical Sciences, Saharanpur, Uttar Pradesh, India
⁵ Joint Director, University Centre of Excellence in Research, Baba Farid University of Health Sciences, Faridkot, Punjab, India

Date of Submission	25-Nov-2020
Date of Decision	01-Mar-2021
Date of Acceptance	25-Mar-2021
Date of Web Publication	17-Aug-2021

Correspondence Address:
Dr. Malika Arora
Research Scientist II, Multidisciplinary Research Unit, Guru Gobind Singh Medical College, Faridkot, Punjab
India

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jihs.jihs_34_20

Abstract

Background: The human intestinal microbiota is very rich in probiotics which includes various species and strains of Lactobacillus, Bifidobacteria, etc. In certain diseased conditions, friendly microbiota may get disturbed or completely eradicated. To make up the loss of such beneficial species, one has to either replenish it from external source or remove the reasons responsible for killing of the microbiota. Similarly, the intestinal microflora also gets disturbed in celiac disease (CD), an autoimmune disorder caused due to the immune-toxic gluten peptides or due to delivery of variety of antibiotics that may act as dysbiotic agents. Aim: The present study was aimed to investigate the replenishment of microbiota after sticking to gluten free diet in celiac disease patient. Materials and Methods: In this study, ten patients each of four groups were included and their faecal samples were collected. Bacterial colonies were isolated from the sample and the colonies were examined by biochemical and morphological profiling. Results: The biochemical and morphological profile of microbiota in faecal samples demonstrated clear-cut regeneration of probiotic species in patients complying with gluten-free diet (GFD). This is the first report on regeneration of microbiota in relation to dietary compliance in CD patients. Conclusion: The results of study may play a great role in detecting the patient compliance to GFD and may serve as a non-invasive prognostic tool replacing biopsy especially for paediatric population.

Keywords: Celiac disease, fecal microbiota, gluten free diet, gut microflora, probiotics

How to cite this article:
Kumari M, Arora M, Kaur N, Singh R, Bansal P. Compliance to gluten-free diet may regenerate probiotic microbiota: First report. J Integr Health Sci 2021;9:8-12

How to cite this URL:
Kumari M, Arora M, Kaur N, Singh R, Bansal P. Compliance to gluten-free diet may regenerate probiotic microbiota: First report. J Integr Health Sci [serial online] 2021 [cited 2023 Jun 10];9:8-12. Available from: https://www.jihs.in/text.asp?2021/9/1/8/323954

Introduction

The human microbiota is the set of microorganisms that colonize within the human body. A plethora of microorganisms reside in the human intestine and these microbes hold a symbiotic relationship with the gut.^[1] Although there is variability of microbiota in the individuals, yet the composition as well as functionality of microbiota is directly associated with the gut health, development of immunity, synthesis of various nutrients, blocking the entry of pathogenic microbes, detoxification of various toxins regenerated inside the body, etc.^[2] Moreover, the gut microbes are considered to be the potential source of novel therapeutic modalities. The gut of an adult is comprised more than 2000 species of variety of bacteria. The microbiota within the gut of a normal healthy subject is mixture of various species and subspecies. In addition, the microbiota is composed of resistant as well as stable ecosystem.^[3] The abundant and diverse microbial community available in each and every microbe is as unique as a fingerprint.^[4] The density and diversity of the microbiota is lying in increasing order from stomach to colon.

The composition of gut microbiota is twisted by both genetic and environmental factors which may keep on changing throughout the lifetime of the individual in continual process.^[5],[6] The characterization of the composition and function of microbiomes from different parts of the body has become feasible due to the availability of various genetic tools and the metagenomic revolution since last one and half decade.^[7] A sound knowledge of composition as well as function of microbiome has led the researchers toward the linking of microbiota to potential diseases, risks, or even to the clear onset of clinical symptoms. Moreover, on the basis of the gut microbiota, scientists across the globe have implicated the use of microbiota in developing disease-specific diagnostics, pathogenesis, and progression of numerous diseases.

In case of healthy individuals, the intestinal microbes are known for multiple functions, but the composition of the microbiota get disturbed in diseases such as diarrhea, antibiotic administration, inflammatory bowel disease, traveller's diarrhea as well as other gastrointestinal disturbances.^[8],[9],[10] Similarly, various studies evidenced the availability of significant alterations of microflora in the patients with celiac disease (CD).^[11],[12] Although the CD is an autoimmune disorder of the small intestine that is caused due to gluten-induced inflammation, it has been observed that species of gut microbiota may activate the inflammatory pathways that trigger celiac reactions.^[13] In addition, it also has been reported that, in case of CD, the intestinal microflora get disturbed as the number of various pathogenic microorganism increases and the number of probiotic species decreases in comparison to healthy adults.^[14] Increase and decrease of some specialized microbes might be either due to the immune-toxic gluten peptides or due to delivery of variety of antibiotics that may act as dysbiotic agents.^[15] It has been considered that microbiota functions in aggregation with the defense system of the body in order to protect the host from pathogen colonization and invasion. The imbalance of the intestinal microflora, i.e., dysbiosis may alter the host immune response and could become a major factor for generating disease and its symptoms.^[16]

Currently, 1%–1.5% of the world population is affected by CD and the only effective treatment for the disease is a strict life-long gluten-free diet. It has been observed that life-long adherence to gluten-free diet (GFD) reverses the disease pathogenesis process as well as changes in the intestinal microflora.^[17] It is pertinent to mention that fecal microbiota is the clear-cut reflection of the gut microbiome; hence, the present study has been designed to study the effect of compliance of GFD on the regeneration of the probiotic species by focusing exclusively on fecal microbiota. Till date, no study on regeneration of microbiota in relation to compliance of GFD has been reported. In order to examine the fact, a pilot study was carried out to investigate the morphological differences in the fecal microbiota of normal human subjects to those who are suffering from CD and are on gluten diet (GD) in contrast to those who are on GFD.

Material and Methods

Enrolment of study subjects: The study subjects will be enrolled to the study as per following classification which is as follows:

Group 1: Normal children attending Out Patient Department (OPD) for immunization clinic
Group 2: Children with known wheat allergy on GFD since 1 year or more were prediagnosed with CD through Tissue Transglutaminase (tTG) and biopsy test
Group 3: Children recently diagnosed with CD (through tTG and biopsy test) but still on GD or wheat diet
Group 4: Children with gastrointestinal abnormality other than gluten allergy.

Informed consent and clinical characterization of the study subjects after the enrolment and identification of study subjects from the tertiary care hospital (GGSMCH), Faridkot written informed consent was obtained from all the parents/guardians of the pediatric patients. Clinical characterization of the study samples was done with respect to their age, gender, clinical symptoms, diagnostic assays used for the detection of allergy, compliance to GFD, etc
Collection of stool sample: Stool samples were obtained from ten children of each group that is; CD children on GFD, CD children not on GFD, gastrointestinal patient, and normal person recruited at Guru Gobind Singh Medical College and Hospital, Faridkot. All the patients were of the age group of 2–14 years CD patients who were included in the study were prediagnosed with CD through tTG and biopsy test. Stool samples from all the included study subjects were collected in sterile plastic containers and stored at −80°C until processing
Processing and screening of fecal sample: One gram of the fecal sample was diluted in De Man, Rogosa and Sharpe (MRS) broth and further used to isolate the probiotic species in fecal sample. One milliliter aliquot of the sample dilutions were spread on preprepared MRS Agar and M16 agar plates (pH 6.2–6.5). The plates were then incubated in anaerobic condition in desiccators for 48–72 h at 37°C. The colonies obtained on the plates were further isolated by streak plate technique and allowed to grow further in the same anaerobic condition. The isolates were then examined by physiological and biochemical characterization. [Figure 1] represents culture and isolation method of pure cultures
Physiological and biochemical characterization: The colonies from fecal sample were subjected to various morphological and biochemical examinations as follows:

Figure 1: Pictorial representation of culture and isolation method of pure cultures

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Gram staining

Isolates from fecal sample were examined for striking differences in the Gram staining pattern. For this, the same samples were subjected to standard Gram staining technique for size, shape, arrangement, and Gram's nature of bacterial samples. The Gram reaction of the isolates was determined by light microscopy after Gram staining. Cultures were grown in appropriate medium at 37°C ± 2°C for 24 h under anaerobic conditions. Cells from fresh cultures were used for Gram staining. The individual colony was picked up aseptically from the agar plate, and a smear was prepared on the slide that was heat fixed followed by staining with crystal violet stain for 1 min and again rinsed with water (5 s). Further decolourizer (95% ethanol) was added for 15–30 s and rinsed with water for 5 s. Finally, saffranine was added for 60–80 s and rinsed with water. Then, under light microscopy, Gram positives and purified isolates were determined. Gram-positive cells will stain purple, while Gram-negative cells stain pink/red.^[18]

Catalase test

Catalase is an enzyme produced by many microorganisms that break down the hydrogen peroxide into water and oxygen and cause gas bubbles. The formation of gas bubbles determines the presence of catalase enzyme and indicates the positive result.^[19]

2H₂O₂ → 2H₂O + O₂

For this purpose, overnight grown cultures of isolates on MRS agar were used. After 24 h, 3% hydrogen peroxide solution was dropped onto randomly chosen colony. The isolates, which did not give gas bubbles, were separately marked and are catalase negative.^[20]

Indole test

The test is done to check the ability of bacteria to break amino acid tryptophan to form indole, pyruvic acid, and ammonia as end products using enzyme tryptophanase. Tryptophanase differentiates indole-positive enterics (e.g., Escherichia coli) from closely related indole-negative enteric.^[21] Ten millilitres tryptophan medium was poured in each McCartney bottle, inoculation processes were done after the media was autoclaved (for 15 min. in 15 lbs pressure at 121°C). Then, the inoculated media (with isolate of fecal sample) were incubated for 24 h at 37°C ± 2°C. E. coli MTCC 4315 was used as a positive control. 0.5 ml of Kovac's reagent was added to the broth and the presence/absence of colored ring was observed.

Oxidase test

The enzyme oxidase, present in certain bacteria, catalyzes the transport of electron from donor bacteria to the redox dye tetra-methyl-p-phenylenediamine dihydrochloride. The dye in the reduced state has a deep purple color. Inoculated plates were incubated at 37°C ± 2°C for 76 h. One colony of each isolates was smeared to filter paper already impregnated with oxidase reagent (1% tetra-methyl-p-phenylenediamine dihydrochloride). Basically, this test is to see if an organism produces cytochrome C oxidase. The positive result was indicated by the production of dark blue color within 7 s.^[22]

Citrate utilization test

This test was done to check the ability of bacteria to utilize sodium citrate as its only carbon source and inorganic NH₄H₂PO₄ as the sole fixed nitrogen source. For this, slants of simmons citrate agar were prepared and inoculated by previously isolated bacterial colonies. The slant was incubated further for 24–28 h at 37°C in anaerobic condition. Appearance of blue color indicates the growth and alkalization.^[23]

Test for probiotic properties

PH tolerance

To determine resistance to stomach, pH 1, 2, and 3 was used in vitro. For this purpose, active cultures (incubated for 16–18 h) were used. The cells were harvested by centrifugation for 10 min at 5000 rpm and 4°C. Pellets were washed once in phosphate-saline buffer (PS at pH 7.2). Then, cell pellets were re-suspended in phosphate-buffered saline (PBS) (pH 3) and incubated at 37°C. Growth was monitored at 0, 1, 2, and 3 h after inoculation, by taking optical density (OD) at 620 nm. Lactobacillus can grow in acidic pH.^[24]

Bile tolerance test

MRS medium containing 0.3% bile (Oxoid) was inoculated with active cultures. Growth was monitored at 0, 1, 2, and 3 h after inoculation, by taking OD at 620 nm.^[24]

Results

Gram staining of isolated colonies for morphological analysis

All the isolates were subjected to Gram staining, and they were examined under light microscope. All the strains gave purple-blue color indicating that all were Gram-positive bacteria. The colonies isolated from CD patients on GFD and that of normal person were found to have similar morphology. The isolated colonies from them were rod shaped. The colonies isolated from CD patients who were not on GFD and those who were having gastrointestinal disease such as diarrhea and blotting had similar morphology. The colonies isolated from them where either spherical or oval in shape. This indicated that CD patients who were on GFD from more than 6 months; their microbiota was restored. [Figure 2] represents morphological profile of microbiota in different study groups.

Figure 2: Morphological profile of microbiota. (a) Gastrointestinal patients. (b) Celiac disease patients not on gluten free diet. (c) Normal control. (d) Celiac disease patients on gluten-free diet

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Catalase test

The result of this test was found to be negative for colonies isolated from fecal sample of CD patient on GFD and normal person while was positive for colonies isolated from fecal sample of CD patient not on GFD and patient with GI tract (GIT) disorders.

Oxidase test

The result showed that colonies isolated from normal person's fecal sample and celiac patients sample who are on GFD were oxidase negative representing the presence of probiotic species in them, while the colonies isolated from celiac patients who were not on GFD, were oxidase positive.

Indole test, methyl red test, and citrate utilization test

These tests also followed the same pattern. These were also negative for colonies isolated from fecal sample of CD patient on GFD and normal person, while was positive for colonies isolated from fecal sample of CD patient on GD and patient with GIT disorders.

Probiotic properties

Resistance to low PH

Being resistant to low pH is one of the major selection criteria for probiotic strains. Since to reach the small intestine, they have to pass through from the stressful conditions of stomach. Although in the stomach, pH can be as low as 1.0, in most of in vitro assays, pH 3.0 has been preferred. Due to the fact that a significant decrease in the viability of strains is often observed at pH 2.0 and below. For selection of the strains resistant to low pH, PBS (pH - 3.0) was used. The time taken during digestion in the stomach is 3 h, so all the isolates were detected whether they were resistant to pH 3.0 during 3 h. After examination of all the isolates that were survived in pH 3.0 were taken to the next step. The isolates from fecal sample of normal person and person on GFD showed resistant to low pH. Other isolates were also resistant, but there OD decreased representing decrease in bacterial colonies. [Figure 3] represents tolerance profile of active cultures isolated from different study groups.

Figure 3: Tolerance profile of active cultures isolated from different study groups

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Tolerance against bile

Isolates resistant to low pH were screened for their ability to tolerate the bile salt. Although the bile concentration of the human gastrointestinal tract varies, the mean intestinal bile concentration is believed to be 0.3% w/v and the staying time is suggested to be 4 h. From the present study results, all of the isolates showed resistant to 0.3% bile salt. [Table 1] represents Comparative biochemical/morphological profile demonstrating the similarity in celiac disease patients on gluten free diet and normal controls to that of gastrointestinal patients and celiac disease patient on gluten diet.

Table 1: Comparative biochemical/morphological profile demonstrating the similarity in celiac disease patients on gluten free diet and normal controls to that of gastrointestinal patients and celiac disease patient on gluten diet

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Discussion

To date, different studies have evidenced an association between intestinal dysbiosis, CD, and gastrointestinal manifestations of the disease. However, no work has been reported yet that defends the regeneration of microbiota in CD patients on GFD. We hypothesized that microbiota could be regenerated if gluten is removed from diet of CD patients. It is pertinent to mention that fecal samples reflect the changes occurring in gut microbiota. Initially, the authors were planning to devise a probiotic-based map using fecal sample that could serve as a futuristic diagnostic modality. While performing the study, the authors observed the regeneration of probiotic species in the fecal samples of the patients complying to GFD. Till date, no study on regeneration of microbiota in relation to compliance has been reported. This study has clarified that if GFD is administered to CD patients for a period of more than 6 months, their microbiota starts regenerating and reverses to normal to a great extent. This study if further carried out can lead to isolation as well as identification of specific missing/regenerating strains using 16s rDNA sequencing technique/bioinformatic tools which could serve as a diagnostic as well as therapeutic modality. In addition, this study will also help in noninvasive monitoring of the compliance of CD patients to check their strict adherence on GFD. Future progress in this area will be yet crucial but highly valuable to provide new clues regarding diagnosis as well as disease management using probiotic microbiome.

Financial support and sponsorship

This study was financially supported by DST-SYST (Project No- SP/YO/097/2017).

Conflicts of interest

There are no conflicts of interest.

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