Using DIAMOND BLASTx, 78,585 and 74,228 contigs were found in ASD and TD groups including complete and incomplete phage domain protein and amino acids, respectively (the data are available upon request). Using Refseq, 1878 and 4774 contigs were identified as phages in ASD and TD groups, respectively (Additional file 2). VirSorter analysis identified 16,772 contigs in ASD and 17,632 contigs in TD in three categories while using Refseq a number of 808 and 1237 contigs were considered as hallmark phages in ASD and TD groups, respectively (Additional file 3), a significantly smaller number of contigs compared to the developed pipeline in the present study. All VirSorter identified contigs were also covered by our pipeline (Additional file 4). The results indicated that in both reference phage (Refseq) and other phage genus (TPA and non-classified phages) are more dominant than the results obtained with other pipelines (at this point Virsorter). The identified functional while ORFs are presented in Additional file 5. The ssDNA phages, Microviridae and Inoviridae for instance, were not identified in neither in ASD nor TD groups. This could be due to two factors, the abundance of ssDNA phages in the gut system [15] and not using viral-specific genome extraction method and library preparation by Dan et al., [11]. The positive associations between fecal dsDNA phages (order Caudovirales) and parameters of the brains’ executive functions have been discussed elsewhere [16]. Therefore, it can be hypothesized that the observed changes in gut phageome could be a potential biomarker for some of the brain performance and behaviors [8].

Mayneris-Perxachs et al. [8] reported correlations between Siphoviridae family and a better executive function and memory in mice. Transplantation of a microbiome enriched with a high level of Siphoviridae phages (> 90%) in mice promoted objects recognition and up-regulated memory-promoting immediate early genes in the prefrontal cortex [8]. On one hand, children affected by different levels of ASD have a poor executive function and memory [17]; and on the other hand the different abundancy of phages in TD and ASD individuals which is observed in the current study, could be a contributing factor of such symptom (Fig. 2A). Hence, there could be link between the prevalence of different genera of phages and brain function. Although the precise roles of phages genera in the microbiome-brain axis are poorly understood [8], their different abundance in ASD compared to TD individuals may result in an enhancement of ASD. Induction of prophage from commensal bacteriome or obtaining new phages from environments e.g., food and direct contact with community might explain the increased level of Caudoviricetes phages [18].

Taxonomic distribution of gut phageome in ASD and TD. A The differential abundance of phage genera detected in ASD and TD. B The differential abundance of different phage species identified in ASD and TD. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 showed a significant difference of the particular phage in ASD with TD. Please refer to Additional File 2 for the detail of differential abundance of the phages

A deeper analysis based on conserved sequences of bacteriophages like phage DNA polymerase and terminase large subunits revealed the presence of enriched phage genera in ASD compared to TD (Fig. 2A). Among the different genera, Mushuvirus (P < 0.001), Brigitvirus (P < 0.001), Toutatisvirus (P < 0.001), Eponavirus (P < 0.001), Taranisvirus (P < 0.001), Wadgaonvirus (P < 0.01), Lughvirus (P < 0.01), Oengusvirus (P < 0.05), Lagaffevirus (P < 0.01), Gemsvirus (P < 0.05), and Efbeekayvirus (P < 0.05) were more abundant in ASD while Punavirus (P < 0.01), Burzaovirus (P < 0.001), Nesevirus (P < 0.001), Peduovirus (P < 0.0001), Pankowvirus (P < 0.001), Lederbergvirus (P < 0.001), Brunovirus (P < 0.001), Oslovirus (P < 0.001), Jahgtovirus (P < 0.001), Hendrixvirinae (P < 0.001), Felsduovirus (P < 0.01), Culoivirus (P < 0.001), and Delmidovirus (P < 0.01) were more abundant in TD (Fig. 2A).

Among the different viral species, Faecalibacterium phages (FP) including Toutatisvirus toutatis (P < 0.01), Mushuvirus mushu (P < 0.0001), Brigitvirus brigit (P < 0.001), Taranisvirus taranis (P < 0. 01), Eponavirus epona (P < 0. 01), Oengusvirus oengus (P < 0. 01), Lagaffevirus lagaffe (P < 0. 01), and Lughvirus lugh (P < 0. 01) had the largest relative abundance in ASD compared to TD (Fig. 2B). The bacterial hosts of these phages, Faecalibacterium spp., mainly represented by Faecalibacterium prausnitzii, are highly presented in the human gut microbiota (5–15% of the human gut microbiome). Those bacteria produce butyrate and other beneficial substances for human health through mechanism such as anti-inflammatory effects or maintaining the Th17/Treg balance [19]. A correlation was observed between the depletion of Faecalibacterium and Crohn’s disease [20], obesity in infants [21] type II, diabetes [22] and aging [23].

In the present study, a high rate of Faecalibacterium phages was observed in ASD compared to TD individuals pointing out a possible role of Faecalibacterium and their prophages in autism. The higher frequency of eight Faecalibacterium phages in ASD individuals could change either the level of Faecalibacterium in the gut (via possible prophage induction and the start of the lytic cycle) or impact the metabolic activities and the function of Faecalibacterium spp. in gut microbiota, or both. The higher abundance of Faecalibacterium spp. in ASD compared to TD individuals, rather than a depletion [11, 24, 25] suggests an impact on metabolic activities instead of the induction of the lytic phase inducing depletion of Faecalibacterium spp. as observed by Cornuault et al. [19], in patients suffering from an inflammatory bowel disease (IBD). The possible roles of prophages on bacterial metabolism mediated by auxiliary metabolic genes (AMGs) were highlighted for some bacteria. For example, a significant increase in middle-chain fatty acids (MCFAs) such as hexanoic acid was observed by Dan et al. [11] in the ASD group. Hexanoic acid can be produced by members of the Clostridium cluster IV and Ruminococcaceae bacterium CPB6 [26]. The Oscillospiraceae family including Faecalibacterium sp. CAG: 74, Subdoligranulum variabile, Clostridium sp. CAG: 269 and Eubacterium sp. CAG: 38 displayed a positive correlation with hexanoic acid level [11]. ASD individuals were associated with higher hexanoic acid levels in the blood in comparison to the TD group [27]. Further investigations are required to disclose the impacts of Faecalibacterium spp. prophages on host metabolism.

In our study, different crAssphages genera (belong to Crassvirales order) were identified in both ASD and TD individuals. For instance,

Blohavirus species (Buchavirus splanchnicus, Buchavirus coli, Blohavirus americanus, and.

Buchavirus hominis) in ASD were significantly abundant ((P < 0. 001) than TD while Buchavirus species (Buchavirus coli, Buchavirus hominis, Buchavirus splanchnicus, Burzaovirus coli and Burzaovirus faecalis) identified in TD were more abundant (P < 0.001) than ASD. Moreover, phages genera of Canhaevirus (Canhaevirus hiberniae), Culoivirus (Culoivirus americanus and Culoivirus intestinalis), Delmidovirus (Delmidovirus intestinihominis), and Jahgtovirus (Jahgtovirus intestinihominis and Jahgtovirus secundus) were identified only in TD indivituals (Fig. 2A and B). This observation was in parallel to the reported low abundance of Bacteroides spp. in ASD by Dan et al. [11]. Regarding the weak associations of Bacteroides with health or disease [28] and the overall high abundance of crAssphages in the human gut virome [29], it could be assumed that crAssphages diversity would be depended with their host (Bacteroides spp.) [29].

To investigate whether ASD individuals display a different gut phageome, a comparison of alpha diversity for phages between ASD and TD groups was performed as well. There was a significant difference between phage Chao1 richness and Shannon’s diversity of the ASD and TD groups (P < 0.0001, Fig. 3A, and P < 0.0001, Fig. 3B, respectively). Based on the alpha diversity, ASD individuals displayed unique gut phage profiles vis-à-vis TD (Fig. 3A and B). Additionally, principal coordinates analysis (PCoA) based on the Bray–Curtis distance between the cases revealed that the gut phageome structure of ASD was different from TD (Fig. 3C). The host-dependent factors such as age, sex, and gastrointestinal symptoms (constipation) were taken into consideration to analyze the phageome in ASD and TD individuals. As shown in Fig. 3D and E, the richness of phages enhanced in older TD individuals (mainly the 7–11 subgroup) compared to the youngest individuals (2–3 years age). However, the ASD subgroups showed no significant differences, neither in Chao1 richness nor in Shannon’s diversity. No additional analysis was performed for sex and gastrointestinal symptoms because the ASD group was composed of only 2 females and all cases suffered from severe constipation (Additional file 1). Dan et al., [11] reported a more heterogeneous and less diverse microbiome in ASD group, and different from the TD group [11]. They also noted that gut microbiota was relatively similar in all ASD age subgroups.

Changes in the gut phageome in ASD and TD individuals. A Chao1 richness and B Shannon’s diversity for the gut phageome of ASD and TD at the contig level. Kruskal–Wallis test, followed by Wilcoxon’s rank-sum test with Bonferroni’s correction were done for alpha diversity between the two test groups. C Principal coordinates analysis (PCoA) was performed based on the Bray–Curtis distance between individual viral groups. D Chao1 richness and E Shannon’s diversity of the gut phageome for TD and ASD according to age from 2 to 11