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BRIEF COMMUNICATION
Year : 2022  |  Volume : 2  |  Issue : 3  |  Page : 91-95

Fecal microbiota in inflammatory bowel disease: Studies of lactobacillus and bifidobacteria diversity


Wellcome Trust Research Laboratory, Department of Gastrointestinal Sciences, Christian Medical College, Vellore, Tamil Nadu, India

Date of Submission06-Jun-2022
Date of Decision08-Jun-2022
Date of Acceptance08-Jun-2022
Date of Web Publication05-Jul-2022

Correspondence Address:
Balakrishnan S Ramakrishna
Department of Gastrointestinal Sciences, Wellcome Trust Research Laboratory, Christian Medical College, Vellore - 632 004, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ghep.ghep_18_22

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  Abstract 


Background: Diversity of microbial species is a hallmark of dysbiosis in inflammatory bowel disease (IBD). This study was undertaken to determine whether the diversity of lactobacilli and bifidobacteria, two regulatory microbial genera, is altered in patients with IBD. Methods: DNA from fecal samples of 20 healthy volunteers, 20 patients with ulcerative colitis (UC), and 25 patients with Crohn's disease (CD) was amplified by the polymerase chain reaction (PCR) for 16S ribosomal RNA gene sequences specific to genus Lactobacillus and Bifidobacterium. Real-time PCR was done to quantitate bacterial abundance. Amplified DNA was also bar coded and sequenced on the Roche 454 platform. Individual operational taxonomic units (OTUs) were taken as sequences with at least 97% similarity. Results: Abundance of Lactobacillus was significantly increased in UC compared to controls; no other difference in Lactobacillus or Bifidobacterium abundance was found between the three groups. Median reads for genus Lactobacillus was similar between control (940), UC (994), and CD (960) groups. The median number of OTUs per sample was not significantly different between controls (6), UC (5.5) and CD (7). The Shannon index of community diversity was similar between controls (median 1.12), UC (median 1.13) and CD (median 1.11). The Simpson D index was also similar between controls (median 0.393), UC (median 0.439) and CD (median 0.409). Median reads for genus Bifidobacterium was similar between control (846), UC (1052), and CD (964) groups. The median number of Bifidobacteria OTUs per sample was similar for controls (6), UC (7) and CD (8.5). The Shannon index of community diversity was similar between controls (median 0.75), UC (median 1.0) and CD (median 1.14). The Simpson D index was also similar between controls (median 0.570), UC (median 0.422) and CD (median 0.409). Conclusion: The abundance of Lactobacillus was increased in UC. Diversity of genus Lactobacillus and Bifidobacterium was not altered in either UC or CD.

Keywords: Bifidobacterium, Crohn's disease, diversity, inflammatory bowel disease, lactobacillus, ulcerative colitis


How to cite this article:
Premkumar K, Ramadass B, Ramakrishna BS. Fecal microbiota in inflammatory bowel disease: Studies of lactobacillus and bifidobacteria diversity. Gastroenterol Hepatol Endosc Pract 2022;2:91-5

How to cite this URL:
Premkumar K, Ramadass B, Ramakrishna BS. Fecal microbiota in inflammatory bowel disease: Studies of lactobacillus and bifidobacteria diversity. Gastroenterol Hepatol Endosc Pract [serial online] 2022 [cited 2022 Aug 14];2:91-5. Available from: http://www.ghepjournal.com/text.asp?2022/2/3/91/349949




  Introduction Top


The role of the gut microbiome in inflammatory bowel disease (IBD) continues to remain a subject for the intense research. In general, it is believed that microbes in the gut lumen influence the presence or absence of inflammation in the gut mucosa.[1],[2],[3] Some microbial species in the gut are characterized as regulatory, influencing the expansion of regulatory T-cells and regulatory B-cells.[4] Other microbial species are characterized as inflammatory, influencing the expansion of inflammatory T-helper cells belonging to Th1, Th2, Th9, and Th17 classes.[4]

Alterations in gut microbiome composition are noted universally in IBD, this state being termed as dysbiosis.[4] Dysbiosis is a frequent occurrence in IBD and may be characterized by one or more of the following phenomena – reduction of bacterial diversity, reduction in number of regulatory species, increase in number of inflammatory species, and reduction of the Firmicutes: Bacteroidetes ratio.

The number of different individual taxa (equivalent to microbial species) within a phylum or class or genus of bacteria is an indicator of the diversity of that particular microbial community. The presence of a greater number of species in an individual is considered to be indicative of greater bacterial diversity of the gut microbiome than the presence of only a few species. Manichanh et al. showed a reduction in the number of ribotypes of certain phyla in Crohn's disease (CD) patients compared to healthy individuals.[5] Reduction in the diversity of specific microbial communities (usually at phylum level) has been demonstrated repeatedly in patients with IBD.[4] We have earlier shown that abundance and diversity of Clostridium leptum, an important carbohydrate fermenting bacterial species represented in phylum Firmicutes, were reduced in patients with IBD compared to controls.[6]

Bifidobacteria and lactobacilli are important regulatory bacteria, many strains of which find use in probiotic food supplements and nutraceuticals because of this important attribute. We could not identify any study that specifically evaluated the species number and diversity of lactobacillus and bifidobacteria in patients with IBD. The present study was undertaken with a view to evaluating this.


  Methods Top


Patients attending the outpatient clinic of the Department of Gastrointestinal Sciences with the following diagnoses were recruited: CD disease (n = 25), ulcerative colitis (UC) (n = 20). CD and UC were diagnosed on the basis of standard criteria,[7],[8] and all patients were in clinical remission at the time of study. Patients attending the outpatient clinic without any evidence of structural gastrointestinal disease (n = 20) were recruited as control subjects. Patients of either gender were included if they were aged 18–70 years. Participants who had received any antibiotics within the past 2 months were excluded from the study. The study was approved by the Institutional Review Board, and written informed consent was obtained from all study participants.

The fresh stool samples obtained from participants were stored at -20°C and batch-processed for DNA extraction using QIAamp DNA stool mini kit (QIAGEN GmbH, Hilden, Germany) as per the manufacturer's instructions, eluted in a final volume of 200 μl and the DNA stored at -20°C. The integrity of DNA was checked by DNA electrophoresis and DNA was quantitated using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

Real-time polymerase chain reaction (PCR) was used to quantify the 16S ribosomal RNA (rRNA) gene specific to Lactobacillus and Bifidobacterium. The primers and PCR conditions have been described in detail in our earlier publications.[9],[10]

For studies of diversity of genus Lactobacillus and Bifidobacterium, fecal DNA from 16 participants in each group was subjected to PCR using bar coded primers to amplify 16S rRNA gene sequences that are conserved for these genera. Lactobacillus specific DNA was amplified using LactoF (TGGAAACAGRTGCTAATACCG) and LactoR (GTCCATTGTGGAAGATTCC) primers to obtain a 232 bp amplicon which contained the hypervariable regions 2 and 3 (V2andV3) of the 16S rRNA gene.[11] This primer set has the ability to detect >98% of all available Lactobacillus sequences. PCR was carried out in 96 well plates with 25–50 ng DNA, ×2.5 hot master mix (5 Prime, Germany), 400nM of each primer (barcoded Forward and Reverse) in 50 μL volume, with initial denaturation at 95°C for 1 min, 30 cycles of denaturation at 95°C for 30 sec, annealing at 50°C for 1 min, and extension at 65°C for 2 min, followed by final extension at 72°C for 10 min. Bifidoacterium DNA was amplified using Bif164-F (GGGTGGTAATGCCGGATG) and Bif662-R (CCACCGTTACACCGGGAA) primers to obtain a 500 bp amplicon which contained hypervariable regions 2 and 3 (V2andV3) of the 16S rRNA gene.[12] This primer set has the ability to detect >98% of all available Bifidobacterium sequences. PCR was carried out in 96 well plates with 25–50 ng DNA, Taq DNA polymerase master mix (1.5 mM and 2.0 mM. Taq DNA Polymerase Master Mix Red is composed of Ampliqon Taq DNA Polymerase, the NH4+ buffer system, dNTPs and magnesium chloride) (Catalog no. 180301 Ampliqon IIII Bioreagents and PCR Enzymes, Denmark), 400nM of each bar coded primer in 50 μL volume, with initial denaturation at 94°C 30 s, 25 cycles of denaturation at 95°C for 30 s, annealing at 54°C for 1 min, and extension at 65°C for 1.5 min, followed by final extension at 72°C for 10 min.

The PCR product, which was 232 bp in size for Lactobacillus and 500 bp for Bifidobacterium, was purified using UltraClean®-htp 96 well PCR clean-up kit (MOBIO Laboratories, Loker Ave West, Carlsbad, CA). DNA free of all reaction components such as primers, enzymes, salts and dNTP's was stored at -20°C and quantified by fluorimetry using SYBR green. PCR purified product yield ranged 1–20 ng/μl. 100 ng of each PCR product were pooled and concentrated by isopropanol method. Pooled amplicons were purified using QIAquick columns (QIAGEN GmbH, Hilden, Germany), and quantitated using Nanodrop 2000. 30 μl of sample sent to Macrogen (Seoul, Korea) for sequencing on the Roche/454 GS Titanium technology platform.

The initial steps in the bioinformatics analysis were done by Macrogen, South Korea. These included quality checking, demultiplexing, and base calling using proprietary software. Paired-end reads were assembled. Reads were clustered into operational taxonomic units (OTUs) sharing 97% sequence identity using UPARSE (Edgar, 2013). The Shannon and Simpson D indices of diversity were calculated, for Lactobacillus and Bifidobacterium species, in each individual.


  Results Top


Participant demographics are shown in [Table 1].
Table 1: Demographics of participants in the study

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Real time polymerase chain reaction studies

[Figure 1] shows the relative abundance of Lactobacillus and Bifidobacterium in relation to total bacteria in the three groups of participants. Bifidobacterium abundance is not significantly different between the three groups, while Lactobacillus was more abundant in UC compared to healthy controls (P < 0.05).
Figure 1: Box and whiskers plot showing the abundance of Lactobacillus (Panel A) and Bifidobacterium (Panel B) in the fecal microbiota of the three groups. Relative difference is a measure of abundance of the 16S rRNA genes relative to the total 16S rRNA gene abundance of universal sequences belonging to Kingdom Bacteria. Box plots show median and interquartile range while whiskers show the range. CD, UC. Lactobacillus was significantly more abundant in UC compared to health controls (P < 0.05). The other differences were not statistically significant. rRNA: Ribosomal RNA, UC: Ulcerative colitis, CD: Crohn's disease

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Sequencing studies

Rarefaction curves obtained were satisfactory [Figure 2].
Figure 2: Representative rarefaction curves indicating that the depth of sequencing was adequate. OTUs: Operational taxonomic units

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Lactobacillus diversity

Median reads for Lactobacillus were similar among the three groups, being 940 for healthy controls, 994 for UC and 966 for CD. An OTU was defined by sequences which shared at least 97% similarity. The median number of Lactobacillus OTUs was 6 in healthy controls, 5.5 in UC patients, and 7 in CD patients. The median Shannon index of community diversity was 1.13 for the entire cohort [Figure 3], suggesting the presence of approximately three equally common species. The median Shannon index was 1.12 for healthy controls, 1.13 for UC patients and 1.11 for CD patients. The median Simpson D index for the entire cohort was 0.398 [Figure 2], indicating that if we pick two microbial populations, randomly from this community there is a nearly 40% chance that they will be similar. The median Simpson index was 0.393 for healthy controls, 0.439 for UC patients and 0.409 for CD patients. The phylogenetic tree reconstructed from the sequences shows that there was no clustering of sequences according to the participant group [Figure 4].
Figure 3: Shannon (left Y axis) and Simpson (right Y axis) diversity indices in each individual participant for the Lactobacillus sequences. V2-V3 hypervariable regions of the 16S rRNA gene specific for genus Lactobacillus were amplified with bar coding and deep sequenced. Microbial community diversity was assessed using the Shannon and Simpson indices. Box plot shows median and interquartile range. None of the differences was statistically significant. rRNA: Ribosomal RNA, UC: Ulcerative colitis, CD: Crohn's disease

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Figure 4: Polar phylogenetic tree reconstructed using the amplified lactobacillus sequences. Controls, CD and UC are marked in different colors. UC: Ulcerative colitis, CD: Crohn's disease

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Bifidobacterial diversity

Median reads for Bifidobacterium were similar between healthy controls, (846) UC 1052) and CD 964). The median number of OTUs per sample was 6 in healthy controls, 7 in UC and 8.5 in CD patients. The median Shannon index was 0.75 for healthy controls, 1.00 for UC patients, and 1.14 for CD patients. The median Simpson D index for the entire cohort was 0.455 [Figure 5], indicating that if we pick two microbial populations randomly from this community there is a 45% chance that they will be similar. The median Simpson index was 0.570 for healthy controls, 0.422 for UC patients, and 0.409 for CD patients. The phylogenetic tree reconstructed from the sequences shows that there was no clustering of sequences according to the participant group [Figure 6].
Figure 5: Shannon (left Y axis) and Simpson (right Y axis) diversity indices in each individual participant for Bifidobacterium sequences. V2-V3 hypervariable regions of the 16S rRNA gene specific for genus Bifidobacterium were amplified with bar coding and deep sequenced. Microbial community diversity was assessed using the Shannon and Simpson indices. Box plot shows median and interquartile range. None of the differences was statistically significant. rRNA: Ribosomal RNA, UC: Ulcerative colitis, CD: Crohn's disease

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Figure 6: Polar phylogenetic tree reconstructed using the amplified Bifidobacterium sequences. Controls, CD and UC are marked in different colors. UC: Ulcerative colitis, CD: Crohn's disease

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  Discussion Top


The present study evaluated the abundance and diversity of lactobacilli and bifidobacterial in patients with UC and CD and concluded that neither abundance nor diversity of these two microbial communities is reduced in IBD.

Manichanh et al. provided the first demonstration of a reduction of microbial diversity in patients with CD.[5] In that study, diversity was reduced at the phylum level, specifically affecting Clostridial clusters in phylum Firmicutes. These bacteria are important carbohydrate fermenters, producing short chain fatty acids as a result of fermentation of unabsorbed carbohydrate in the colon. Multiple other studies since then have shown reduction in the abundance of bacteria belong to phylum Firmicutes in the fecal microbiota[4] and in mucosa-associated microbiota.[13]

Despite the number of studies investigating dysbiosis in IBD, there is no information on specific changes in bacteria belong to genus Lactobacillus or Bifidobacterium in IBD. Bacteria belonging to these two genera are often used as probiotics, because of specific immunomodulatory attributes that they are believed to possess. These bacteria attenuate experimental colitis in animal models.[14] However, in this study, we did not find any reduction in abundance or diversity of bacteria belonging to either genus. Interestingly, Lactobacillus abundance was increased in patients with UC, consistent with our studies of mucosa-associated microbiota in IBD, and consistent with findings by other investigators.[15]

The limitation of the present study is the small number of participants per group, and the fact that we did not specifically sequence the entire gut microbiome in these individuals. The latter was due to the fact that the study was specifically target to evaluate diversity at genus level. Diversity was calculated based on classifying OTUs sharing 97% sequence similarity, a method that has some limitations.[16] It is possible to speculate that in UC there may an increase in certain species or strains of Lactobacillus that may be pathogenic rather than protective.


  Conclusion Top


Diversity of lactobacilli and bifidobacteria was not altered in either UC or CD compared to controls.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Manichanh C, Rigottier-Gois L, Bonnaud E, Gloux K, Pelletier E, Frangeul L, et al. Reduced diversity of faecal microbiota in Crohn's disease revealed by a metagenomic approach. Gut 2006;55:205-11.  Back to cited text no. 5
    
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Kabeerdoss J, Sankaran V, Pugazhendhi S, Ramakrishna BS. Clostridium leptum group bacteria abundance and diversity in the fecal microbiota of patients with inflammatory bowel disease: A case-control study in India. BMC Gastroenterol 2013;13:20.  Back to cited text no. 6
    
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Ramakrishna BS, Makharia GK, Abraham P, Ghoshal UC, Jayanthi V, Agarwal BK, et al. Indian society of gastroenterology consensus on ulcerative colitis. Indian J Gastroenterol 2012;31:307-23.  Back to cited text no. 7
    
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Ramakrishna BS, Makharia GK, Ahuja V, Ghoshal UC, Jayanthi V, Perakath B, et al. Indian society of gastroenterology consensus statements on crohn's disease in India. Indian J Gastroenterol 2015;34:3-22.  Back to cited text no. 8
    
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Balamurugan R, Janardhan HP, George S, Raghava MV, Muliyil J, Ramakrishna BS. Molecular studies of fecal anaerobic commensal bacteria in acute diarrhea in children. J Pediatr Gastroenterol Nutr 2008;46:514-9.  Back to cited text no. 9
    
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Balamurugan R, Janardhan HP, George S, Chittaranjan SP, Ramakrishna BS. Bacterial succession in the colon during childhood and adolescence: Molecular studies in a Southern Indian village. Am J Clin Nutr 2008;88:1643-7.  Back to cited text no. 10
    
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Byun R, Nadkarni MA, Chhour KL, Martin FE, Jacques NA, Hunter N. Quantitative analysis of diverse Lactobacillus species present in advanced dental caries. J Clin Microbiol 2004;42:3128-36.  Back to cited text no. 11
    
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Hopkins MJ, Macfarlane GT, Furrie E, Fite A, Macfarlane S. Characterisation of intestinal bacteria in infant stools using real-time PCR and northern hybridisation analyses. FEMS Microbiol Ecol 2005;54:77-85.  Back to cited text no. 12
    
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Kabeerdoss J, Jayakanthan P, Pugazhendhi S, Ramakrishna BS. Alterations of mucosal microbiota in the colon of patients with inflammatory bowel disease revealed by real time polymerase chain reaction amplification of 16S ribosomal ribonucleic acid. Indian J Med Res 2015;142:23-32.  Back to cited text no. 13
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14.
Nanda Kumar NS, Balamurugan R, Jayakanthan K, Pulimood A, Pugazhendhi S, Ramakrishna BS. Probiotic administration alters the gut flora and attenuates colitis in mice administered dextran sodium sulfate. J Gastroenterol Hepatol 2008;23:1834-9.  Back to cited text no. 14
    
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Wang W, Chen L, Zhou R, Wang X, Song L, Huang S, et al. Increased proportions of Bifidobacterium and the Lactobacillus group and loss of butyrate-producing bacteria in inflammatory bowel disease. J Clin Microbiol 2014;52:398-406.  Back to cited text no. 15
    
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Nguyen NP, Warnow T, Pop M, White B. A perspective on 16S rRNA operational taxonomic unit clustering using sequence similarity. NPJ Biofilms Microbiomes 2016;2:16004.  Back to cited text no. 16
    


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