Isolation and characterisation of ΦcrAss002, a crAss-like phage from the human gut that infects Bacteroides xylanisolvens

Abstract

Background: The gut phageome comprises a complex phage community of thousands of individual strains, with a few highly abundant bacteriophages. CrAss-like phages, which infect bacteria of the order Bacteroidales, are the most abundant bacteriophage family in the human gut and make an important contribution to an individual's core virome. Based on metagenomic data, crAss-like phages form a family, with four sub-families and ten candidate genera. To date, only three representatives isolated in pure culture have been reported: ΦcrAss001 and two closely related phages DAC15 and DAC17; all are members of the less abundant candidate genus VI. The persistence at high levels of both crAss-like phage and their Bacteroidales hosts in the human gut has not been explained mechanistically, and this phage-host relationship can only be properly studied with isolated phage-host pairs from as many genera as possible.

Results: Faeces from a healthy donor with high levels of crAss-like phage was used to initiate a faecal fermentation in a chemostat, with selected antibiotics chosen to inhibit rapidly growing bacteria and selectively enrich for Gram-negative Bacteroidales. This had the objective of promoting the simultaneous expansion of crAss-like phages on their native hosts. The levels of seven different crAss-like phages expanded during the fermentation, indicating that their hosts were also present in the fermenter. The enriched supernatant was then tested against individual Bacteroidales strains isolated from the same faecal sample. This resulted in the isolation of a previously uncharacterised crAss-like phage of candidate genus IV of the proposed Alphacrassvirinae sub-family, ΦcrAss002, that infects the gut commensal Bacteroides xylanisolvens. ΦcrAss002 does not form plaques or spots on lawns of sensitive cells, nor does it lyse liquid cultures, even at high titres. In keeping with the co-abundance of phage and host in the human gut, ΦcrAss002 and Bacteroides xylanisolvens can also co-exist at high levels when co-cultured in laboratory media.

Conclusions: We report the isolation and characterisation of ΦcrAss002, the first representative of the proposed Alphacrassvirinae sub-family of crAss-like phages. ΦcrAss002 cannot form plaques or spots on bacterial lawns but can co-exist with its host, Bacteroides xylanisolvens, at very high levels in liquid culture without impacting on bacterial numbers. Video abstract.

Keywords: Bacteriophages; Human gut phageome; Human microbiome; Phage-host interactions; crAss-like phages; crAssphage.

Fig. 1

The effect of antibiotic selective enrichment in faecal fermenter on the abundance of bacterial orders and the parallel effect on different crAss-like phage abundances. a The mean (across three experimental runs) relative abundances of the key bacterial orders under both conditions tested (only orders with relative abundance of 1% in any of the samples are shown). b The mean relative abundance of crAss-like phage contigs per genus as a percentage of total crAss-like reads. The crAss-like phage contigs are coloured based on candidate genus. CrAss-like phages that resolve into five of the ten crAss-like family candidate genera were detected. c Absolute quantification of each crAss-like phage detected using qPCR with phage or genus-specific primers targeting a segment of the terminase or primase gene. The error bars indicate standard deviation (n = 3). The asterisks denote statistical significance in p value (*p value ≤ 0.01; ** p value ≤ 0.001)

Fig. 2

Circular genome map of the ΦcrAss002 genome. The innermost ring (blue; positive strand, green; negative strand) depicts G + C skew, the central ring (black) shows G + C content and the outer ring (red) highlights Illumina read coverage along the genome. The outermost circle shows coding genes (CDS) which are labelled on HHpred function predictions. CDS are coloured based on general function which corresponds to the legend. No function predictions were possible for genes which are unlabelled

Fig. 3

In silico characterisation of ΦcrAss002. a To examine relatedness of ΦcrAss002 with twenty other phages of candidate genus IV identified in silico, a heatmap was generated based on average nucleotide identity (ANI). b Whole genome comparisons of ΦcrAss002 and a subset of related phages to highlight synteny and genome organisation. In decreasing order from the top are phages with higher to lower ANI/relatedness. Genes with predicted functions are colour-coded based on generalised function. Areas of tBLASTx homology between the genomes are highlighted. c Whole genome comparison of ΦcrAss002 with ΦcrAss001 (sequence from pure isolate) and the prototypical crAssphage (sequence solely in silico) to examine synteny and homology. Regions of homology (tBLASTx) are highlighted

Fig. 4

Phylogenetic tree of major capsid proteins encoded by published complete genomes of crAss-like phages. Protein sequences were aligned using MUSCLE, approximately-maximum-likelihood phylogenetic trees were generated using FastTree. Branch support values calculated using SH-test. Tree tip colours correspond to candidate genera as proposed in [19]. ΦcrAss002 label is highlighted in red. Previously well-characterised uncultured phage genomes and cultured isolates are highlighted in boldface font. Accession numbers of genomes in NCBI GenBank/RefSeq/WGS databases are provided

Fig. 5

Biological characterisation of ΦcrAss002. a Transmission electron micrograph, generated from ΦcrAss002 enriched lysate, stained with uranyl acetate. Micrographs show podovirus-like virions with a diameter of ~77 nm and a simple tail structure. Scale bars represent 100 nm. b In vitro propagation of ΦcrAss002 over 11 days. Titre quantification was performed using qPCR. c Investigation of the ability of ΦcrAss002 to propagate on commercial B. xylanisolvens DSM18836 and the only other confirmed crAss-like phage host to date, B. intestinalis 919/174 via liquid propagation over 5 days. Error bars indicate standard deviation (n = 3)

Fig. 6

Continuous co-culture of ΦcrAss002 and Bacteroides xylanisolvens APCS1/XY. a Serial co-culturing of ΦcrAss002 over 30 days. b Serial propagation of the phage on naïve host cells (absent of phage exposure for ~10 generations). c Equivalent experiment using ten B. xylanisolvens cultures with recent phage exposure. ΦcrAss002 titre is shown in copies/ml, determined by absolute qPCR. Statistical analysis was performed by the one-way ANOVA (p < 0.001) with Tukeys as post-test comparing titres. A statistical significance of the difference between the lowest titre and the highest titre sustained shown (p < 0.001***). The arrows indicate the approximate titre ΦcrAss002 at the initiation of each propagation cycle. The error bars represent standard deviation (n = 3)

Fig. 7

The impact of ΦcrAss001 and ΦcrAss002 on host counts in a defined bacterial community. Continuous fermentations were performed in parallel with and without phage addition. Respective hosts were included B. xylanisolvens (ΦcrAss002) and B. intestinalis (ΦcrAss001). Absolute quantification, by qPCR, was performed on total DNA. a Absolute quantification of the bacterial community structure without phage addition. b Equivalent graph showing the community structure in the presence of the phages. c The titre and propagation dynamic of ΦcrAss002. d Equivalent graph for ΦcrAss001. The error bars represent standard deviation (n = 3)