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CORRESPONDENCE
Year : 2014  |  Volume : 32  |  Issue : 3  |  Page : 347-348
 

Metagenomic analysis of diarrheal stool samples of HIV infected individual and HIV-uninfected individual using 16SrDNA sequencing


1 Division of Microbiology and Clinical Pathology, National AIDS Research Institute, Bhosari, Pune, Maharashtra, India
2 Department of Biotechnology, Saint Berchman's College, Changanacherry, Kerala, India

Date of Submission13-Jun-2013
Date of Acceptance17-Dec-2013
Date of Web Publication10-Jul-2014

Correspondence Address:
V Nema
Division of Microbiology and Clinical Pathology, National AIDS Research Institute, Bhosari, Pune, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.136606

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How to cite this article:
Nema V, Nair R. Metagenomic analysis of diarrheal stool samples of HIV infected individual and HIV-uninfected individual using 16SrDNA sequencing. Indian J Med Microbiol 2014;32:347-8

How to cite this URL:
Nema V, Nair R. Metagenomic analysis of diarrheal stool samples of HIV infected individual and HIV-uninfected individual using 16SrDNA sequencing. Indian J Med Microbiol [serial online] 2014 [cited 2019 Sep 23];32:347-8. Available from: http://www.ijmm.org/text.asp?2014/32/3/347/136606


Dear Sir,

The role of microflora have been explored in healthy individuals and individuals with various metabolic dysfunctions like obesity and others. [1],[2],[3] However, the putative ability of certain bacterial species to enhance gut health, and the practicality of intervention strategies such as the use of prebiotics to achieve this, remains uncertain and poorly understood in case of various infections including human immunodeficiency virus (HIV) infection. Hence screening bacterial communities and other parasites will be of great utility to understand the interaction and will guide us to scale up our future efforts towards an integrated management of HIV-infected individuals. This pilot study was an effort to explore the feasibility of such analysis in finding indications about microbial roles in infection and its progression. In the present experiment, the gut microbiota was studied and compared in two stool samples.

Leftover samples from a previously completed study were obtained after the removal of identifiers with the permission of institutional ethics committee. Sample 1 was from a HIV-positive individual (Male/27Yr) with a CD4 count of 930 cells per microlitre, whereas sample 2 was from an uninfected individual (Male/25Yr). Both the samples were diarrheal samples. Samples were processed as described earlier. [4] Segregated colonies obtained from the cloning reactions from both the samples from infected and non-infected individuals were picked and 40/46 and 21/32 clones were found positive by PCR performed to check for insert [Figure 1]a]. For sequencing reactions, plasmids isolated from 30 and 20 positive clones from sample 1 and 2, respectively, were used [Figure 1]b]. Analysis revealed the close similarities of the amplified 16SrRNA gene sequences with microbial genera including  Escherichia More Details, Dialister, Lactobacillus, Prevotella, Bacteroides, and a few uncultured and unidentified bacteria in sample 1 whereas sample 2 showed a less diverse population with genera like Escherichia, Streptococcus and Lactobacillus. Unique sequences were submitted to GenBank [GenBank: HQ318717, GenBank: HQ318718, GenBank: HQ318719, GenBank: JQ627622, GenBank: JQ627623 and GenBank: JQ627624].
Figure 1: (a) Amplification of inserted gene in clones [Amplicon at around 1800bp (1769bp)] (b) Plasmid isolation from positive clones

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The kind of flora observed in two samples showed a different makeup. HIV-infected individual harboured a slightly diverse flora in comparison to what an uninfected individual showed. The species indicated by sequencing in the uninfected individual mostly reveals the normal flora. Sequences from the HIV-infected sample indicated the presence of multiple sequences similar to various species of Dialister, Lactobacillus, Bacteroides and Prevotella. Although present in the gut of healthy adults, Dialister species are normally associated with the oral cavity and have been linked to opportunistic infections when found outside the oral cavity. [5]

The potential links between viruses, microbiota, and the host immune system have been explored and it was found that HIV-1 patients have between 10- and 10000-fold more opportunistic pathogens in the intestine, and a reduced number of commensal bacteria. [6] Ellis et al. used molecular techniques to classify bacteria in stool of HIV-infected patients beginning ART and HIV-negative controls. Their preliminary results suggest that compared to controls, there are substantial differences in the composition of gut flora in HIV-infected patients, with an increased prevalence of certain bacterial orders with high LPS content and greater TLR-stimulating potential. [6] This is a crucial observation that supports the objective of the present pilot study and encourages more studies like this. HIV being a complex multi-factorial disease requires multi-pathway understanding and multi-targeting approaches. Hence, understanding microbial roles and considering its applications based on healthy constitutions become as important as understanding the pathogen and host immune system.

 
 ~ References Top

1.Human Microbiome Project Consortium. Structure, function and diversity of the healthy human microbiome. Nature 2012;486:207-14.  Back to cited text no. 1
[PUBMED]    
2.Turnbaugh PJ, Backhed F, Fulton L, Gordon JI. Diet-induced obesity is linked to marked but reversible alterations in the mouse distal gut microbiome. Cell Host Microbe 2008;3:213-23.  Back to cited text no. 2
    
3.Spencer MD, Hamp TJ, Reid RW, Fischer LM, Zeisel SH, Fodor AA. Association between composition of the human gastrointestinal microbiome and development of fatty liver with choline deficiency. Gastroenterology 2011;140:976-86.  Back to cited text no. 3
    
4.Nema V. Rectification of artificial molecular recombination with the use of high fidelity enzyme in the amplification of 16S rDNA sequences from stool sample. All Res J Biol 2012;3:6-9.  Back to cited text no. 4
    
5.Siqueira JF Jr, Rocas IN. Uncultivated phylotypes and newly named species associated with primary and persistent endodontic infections. J Clin Microbiol 2005;43:3314-9.  Back to cited text no. 5
    
6.Ellis CL, Ma ZM, Mann SK, Li CS, Wu J, Knight TH, et al. Molecular characterization of stool microbiota in HIV-infected subjects by panbacterial and order-level 16S ribosomal DNA (rDNA) quantification and correlations with immune activation. J Acquir Immune Defic Syndr 2011;57:363-70.  Back to cited text no. 6
    


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