Metabarcoding analysis of eukaryotic microbiota in the gut of HIV-infected patients
Authors: Ibrahim Hamad
Isabelle Ravaux
Rita Abou Abdallah
Caroline Michelle
Andreas Stein
Jean-Christophe Lagier
Fadi Bittar
Didier Raoult
Hervé Tissot-Dupont
Saadia Mokhtari
Journal: PLoS ONE (2018)
Discipline: Multidisciplinary
Keywords: gut microbiota; eukaryotic microbiota; HIV-infected patients; DNA extraction protocols; clone sequencing
Affiliations: Aix-Marseille Université, CNRS 7278, IRD 198, Inserm 1095, AP-HM, URMITE, IHU Méditerranée Infection, Marseille, France., Charmo University, Charmo Research Center, Chamchamal/Sulaimani, Iraq., Service de Maladies Infectieuses et tropicales, CHU de la Conception, IHU Méditerranée Infection, Marseille, France., Assistance Publique Hôpitaux de Marseille, CHU Nord, Pôle Infectieux, IHU Méditerranée Infection, Marseille, France.

Abstract:  Research on the relationship between changes in the gut microbiota and human disease, including AIDS, is a growing field. However, studies on the eukaryotic component of the intestinal microbiota have just begun and have not yet been conducted in HIV-infected patients. Moreover, eukaryotic community profiling is influenced by the use of different methodologies at each step of culture-independent techniques. Herein, initially, four DNA extraction protocols were compared to test the efficiency of each method in recovering eukaryotic DNA from fecal samples. Our results revealed that recovering eukaryotic components from fecal samples differs significantly among DNA extraction methods. Subsequently, the composition of the intestinal eukaryotic microbiota was evaluated in HIV-infected patients and healthy volunteers through clone sequencing, high-throughput sequencing of nuclear ribosomal internal transcribed spacers 1 (ITS1) and 2 (ITS2) amplicons and real-time PCRs. Our results revealed that not only richness (Chao-1 index) and alpha diversity (Shannon diversity) differ between HIV-infected patients and healthy volunteers, depending on the molecular strategy used, but also the global eukaryotic community composition, with little overlapping taxa found between techniques. Moreover, our results based on cloning libraries and ITS1/ITS2 metabarcoding sequencing showed significant differences in fungal composition between HIV-infected patients and healthy volunteers, but without distinct clusters separating the two groups. Malassezia restricta was significantly more prevalent in fecal samples of HIV-infected patients, according to cloning libraries, whereas operational taxonomic units (OTUs) belonging to Candida albicans and Candida tropicalis were significantly more abundant in fecal samples of HIV-infected patients compared to healthy subjects in both ITS subregions. Finally, real-time PCR showed the presence of Microsporidia, Giardia lamblia, Blastocystis and Hymenolepis diminuta in different proportions in fecal samples from HIV patients as compared to healthy individuals. Our work revealed that the use of different sequencing approaches can impact the perceived eukaryotic diversity results of the human gut. We also provide a more comprehensive view of the eukaryotic community in the gut of HIV-infected patients through the complementarity of the different molecular techniques used. Combining these various methodologies may provide a gold standard for a more complete characterization of the eukaryotic microbiome in future studies. 

Retracted: No

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 The Japan-US seminar paper was submitted to be included in a special section of the Nuclear Engineering and Design journal. The papers are intended to be the same, and the preface to that section notes u201cFollowing the Seminar, six papers were submitted, reviewed and accepted for publication in Nuclear Engineering and Designu201d; this paper was among them. Differences between the conference and journal papers are attributable to refinement and editorial changes to make it suitable for the journal. ,