BACKGROUND: Lactiplantibacillus plantarum HEAL9 and Lacticaseibacillus paracasei 8700:2 positively affect the fecal bacteriome in children with celiac disease autoimmunity after 6 months of supplementation. The aim of the present investigation was to study the effects of Lactiplantibacillus plantarum HEAL9 and Lacticaseibacillus paracasei 8700:2 on the single-cell parasitome, with a primary focus on Blastocystis. METHODS: Stool samples were collected from 78 Swedish children with celiac disease autoimmunity participating in a randomized, double-blind, placebo-controlled clinical trial to either receive a mixture of supplementation with L. plantarum HEAL9 and L. paracasei 8700:2 (n = 38) or placebo (n = 40). A total of 227 stool samples collected at baseline and after 3 and 6 months of intervention, respectively, were retrospectively analyzed for Blastocystis by quantitative real-time PCR and subtyped by massively parallel amplicon sequencing. Other single-cell parasites were detected by untargeted 18S rDNA amplicon sequencing and verified by real-time PCR. The relation between the parasites and the bacteriome community was characterized by using 16S rDNA profiling of the V3-V4 region. RESULTS: Three different single-cell protists were identified, of which the highest prevalence was found for Dientamoeba fragilis (23.1%, 18/78 children), followed by Blastocystis (15.4%, 12/78) and Entamoeba spp. (2.6%, 2/78). The quantity of the protists was stable over time and not affected by probiotic intervention (P = 0.14 for Blastocystis, P = 0.10 for D. fragilis). The positivity of the protists was associated with increased bacteriome diversity (measured by multiple indices, P < 0.03). Bacterial composition was influenced by the presence of the protists: positivity of Blastocystis was inversely associated with Akkermansia (at the levels of the genus as well as its family, order, class and phylum); P < 0.002), Faecalibacterium (P = 0.003) and Romboutsia (P = 0.029); positivity of D. fragilis was inversely associated with families Enterobacteriaceae (P = 0.016) and Coriobacteriaceae (P = 0.022) and genera Flavonifractor (P < 0.001), Faecalibacterium (P = 0.009), Lachnoclostridium (P = 0.029), Ruminococcus (P < 0.001) and Granulicatella (P = 0.018). CONCLUSIONS: The prevalence of single-cell protists is low in children with celiac disease autoimmunity. The colonization was stable regardless of the probiotic intervention and associated with increased diversity of the fecal bacteriome but inversely associated with some beneficial bacteria.

Celiac Disease and Diabetes Unit Department of Clinical Sciences Lund University Malmö Sweden

Department of Food Technology Engineering and Nutrition Lund University Lund Sweden

Department of Medical Microbiology 2nd Faculty of Medicine Charles University Prague Czechia

Department of Paediatrics 2nd Faculty of Medicine Charles University Prague Czechia

Institute of Parasitology Biology Centre Czech Academy of Sciences České Budějovice Czechia

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