Sohail et al. Gut Pathog (2021) 13:21 https://doi.org/10.1186/s13099-021-00411-x Gut Pathogens SHORT REPORT Open Access Microbiome profling of rotavirus infected children sufering from acute gastroenteritis Muhammad U. Sohail1, Hebah A. Al Khatib2, Asmaa A. Al Thani2,3, Khalid Al Ansari4, Hadi M. Yassine2,3* and Maha Al‑Asmakh2,3,5* Abstract Background: Rotavirus (RV) is a leading cause of pediatric diarrhea and mortality worldwide. The virus causes acute gastroenteritis characterized by moderate to severe vomiting, diarrhea, dehydration, and fever. Microbial dysbiosis caused by RV infection may signifcantly infuence disease prognosis and the development of other chronic diseases. The gut microbiome plays a vital role in enteric immune response for rotavirus vaccine (RVV) that requires further elucidations. The current study evaluates the gut microbiome of RV positive children and compares gastroenteritis manifestation in children admitted to the Pediatric Emergency Centre, Hamad Medical Cooperation, Doha, Qatar. Stool samples were collected from thirty‑nine RV positive and eight healthy control children. 16S rRNA sequence was performed using the Illumina MiSeq platform. Results: The data demonstrated a signifcant increase in microbiome diversity denoted by higher relative abun‑ dances of phylum Proteobacteria (p 0.031), Fusobacteria (p 0.044) and genus Streptococcus (p 0.001) in the infected group relative to the control.= Similarly, district clustering= pattern (PERMANOVA p 0.01) and≤ higher species richness (Shannon entropy p 0.018) were observed in the children who received two RVV= doses compared with the non‑vaccinated or single‑dose= groups. These microbiome changes were represented by over‑abundance of phylum Bacteroidetes (p 0.003) and Verrucomicrobia (p 0.001), and lower expression of family Enterobacteriaceae in two RVV doses group.= However, microbiome composition≤ was not associated with diarrhea, vomiting, and other param‑ eters of gastroenteritis. Conclusions: The observations assert signifcant microbial signatures of RVV, which is dose‑dependent, and suggest manipulating these microbes as a novel approach for improving RVV efcacy. Further studies are warranted to investi‑ gate the immune status of these patients and mechanistic investigation to enhance RVV seroconversion. Keywords: Rotavirus, Rotavirus vaccine, Acute gastroenteritis, Microbiome, Proteobacteria Background deaths in children and was responsible for an estimated Diarrhea is the second leading cause of mortality in 258 million infectious diarrhea cases worldwide [1]. children after pneumonia, causing one out of nine child Despite the availability of the rotavirus vaccine (RVV), deaths worldwide [1]. Te Global Burden of Disease the virus is the leading cause of diarrhea-related mor- Study 2016 estimated that rotavirus (RV) caused 128,500 talities in children under fve years of age [2]. Literature shows that vaccine efcacy is region-specifc and depicts *Correspondence: [email protected]; [email protected] poor seroconversion, particularly in low-and middle- 3 Department of Biomedical Sciences, College of Health Sciences, QU income countries [2]. Human clinical trial data suggest a Health, Qatar University, P.O. Box 2713, Doha, Qatar possible link between the gut microbiome and the enteric 5 Biomedical and Pharmaceutical Research Unit, QU Health, Qatar University, P.O. Box 2713, Doha, Qatar immune system’s response for low RVV efcacy [2–4]. Full list of author information is available at the end of the article © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Sohail et al. Gut Pathog (2021) 13:21 Page 2 of 9 Te human gut is occupied by a large and complex indexes, and other demographic information are pre- microbial community that maintains the physiological sented in Table 1. homeostasis of the host immune and neuroendocrine systems. Te gastrointestinal microbiome is nvolved in the digestion and absorption of nutrients and is essiential Amplicon sequencing for priming the gut-associated lymphoid tissue (GALT) Genomic DNA was extracted using QIAamp DNA Mini that helps inhibit local or systemic infections. Terefore, Stool Kit (cat. no. 51,504; Qiagen, Hilden, Germany). many gut realted disorders including viral infections have DNA quality control (QC) was checked using Nan- been associated with changes in the microbiome [5, 6]. oDrop-2000 (Termo Fisher Scientifc, USA), Qubit-4 Te gut microbiome has been implicated in the patho- (Life Technologies, USA), and Agilent bioanalyzer 2100 genesis of several viral pathogens, including adenovirus, (Agilent Technologies, US). DNA libraries were prepared astrovirus, calicivirus, coronavirus, norovirus, polio- using 337F/805R primer-pair targeting V3-V4 region of virus, and RV [7, 8]. Furthermore, mounting evidence the 16S rRNA gene. All libraries were dual indexed using also supports the role of gut microbiome in determining Illumina Nextera XT Library Prep. Kit (FC-131-1002, immunogenicity and seroconversion of the oral RVV [2, Illumina Inc., USA). Amplicon libraries were normalized 3, 6]. Germ-free murine studies have shown that normal using Agencourt AMpure XP beads (Beckman Coulter, GALT development requires bacterial colonization of the USA). All libraries were pooled together for performing gut with specifc microbes that increase IgA secretion sequencing using MiSeq v3 kit (MS-102-3003; Illumina and enhance RVV immunogenicity [9]. Murine studies Inc., USA). MiSeq run generated 300 bp longpaired-end also suggest that the gut microbiome can help prevent reads implemented as FASTQ fles. and cure RV infection and serve as an adjuvant in RVV vaccination, as demonstrated by the ability of segmented Data analysis flamentous bacteria to cure RV by increasing epithelial cell turnover and antibody response [10]. MiSeq data were analyzed using QIIME2 software. In Compared to the healthy controls, children infected brief, FASTQ fles were imported into QIIME2 artifacts. with RV have reduced microbiome diversity, and antibi- Sequence QC was performed using DADA2 plugin to otic treatment further exacerbates microbiome diversity generate FeatureTable [Frequency] and FeatureData and disease outcomes [3, 6]. Nevertheless, the available [Sequence]. Phylogenetic diversity analysis was per- literature on the involvement of gut microbiome in the formed using maft-fasttree plugin. Greengenes 13 − 8 pathogenesis of RV infection is still scarce, underlining database was used for taxonomic analysis in q2-feature- the need for further investigation. Terefore, the present classifer. Alpha and beta-diversity analysis was per- study investigates gut microbiome composition in chil- formed at 13,813 bp sampling depth using Shannon dren sufering from RV infection and correlates microbi- and weighted_unifrac indexes, respectively. Taxonomic ome composition and diversity with gastroenteritis.‎ description for microbiome was only represented at phylum and species/genus level. Statistical analysis was performed on only those phyla and species/genera with Materials and methods a relative abundance of at least 0.5 % or was present in Patient sampling at least 50 % of the samples, and the remaining bacterial Stool samples were collected from children afected by clades were discarded. acute gastroenteritis (AGE) during their visit to the Pedi- atric Emergency Centre (PEC), HMC, Doha, Qatar. All collected samples were screened using the FilmArray Statistical analysis ® Gastrointestinal (GI) Panel Kit (BioFire Diagnostics, Te participants were classifed into multiple categorical USA) for viral and bacterial infections. Multiple clini- groups based on their history of infection (healthy and cal characteristics of AGE were present in the admitted sick), vaccine history, the RV genotype, gender, age, and children, including fever, diarrhea, vomiting, and dehy- severity index of vesicular gastroenteritis. Non-paramet‎ - dration. Vesikari score system was used to evaluate the ric Mann-Whitney U or Kruskal Wallis tests were con- severity of AGE of the participants [11]. Similarly, chil- ducted to compare microbiome taxonomy and diversity dren were also categorized based on their vomiting and among diferent categorical groups. Principal‎ coordinates diarrhea indexes. Te vomiting and diarrhea indexes (PCoA) plots were constructed for beta diversity analy- were calculated by multiplying their frequencies with sis, and statistical analyses were performed using pair- durations in days (vomiting/diarrhea frequency × dura- wise