Protocol for the isolation, sequencing, and analysis of the gut phageome from human fecal samples

Bikel, Shirley; Gallardo-Becerra, Luigui; Cornejo‐Granados, Fernanda; Ochoa-Leyva, Adrián

doi:10.1016/j.xpro.2022.101170

Cited by 6 publications

(6 citation statements)

References 21 publications

(25 reference statements)

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“…Until comprehensive genome-/ or proteome-based taxonomic phage reference catalogues are available, k-mer-based nucleotide database-dependant matching may extent the scope of applying metagenomics to studying the human gut phageome, commonly referred to as "known unknown" or "viral dark matter" component of the gut microbiome. 8,10, 15,18 Consistent with the dominance observed in previous human gut phageomes [12][13][14] , dsDNA phages of the Caudovirales order (mainly Siphoviridae) dominated the gut phageome in cachectic and non-cachectic patients. The human gut phageome, however, is not yet fully characterized 19 .…”

Section: Discussionsupporting

confidence: 63%

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Lower abundance of human gut virus species is associated with cancer cachexia

Stang

Weilert

Illig

et al. 2022

Preprint

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Cancer cachexia has been linked to gut bacterial alterations, but alterations of gut viruses, mostly bacteriophages, have not yet been explored. We performed shotgun metagenomic sequencing of DNA from stool samples of 78 cachectic and 42 non-cachectic cancer patients. K-mer-based matching to reference databases revealed abundance variations of bacteria and viruses. Beyond bacterial alterations, cachectic patients exhibited significantly lower bacteriophage abundance, predominantly affecting Caudovirales and Siphoviridae species (double-stranded DNA) but also Inoviridae and Microviridae families (single-stranded DNA). Machine learning models exploiting the data for classification between cachectic and non-cachectic state yielded an AUC of 0.704. Caudovirales and Siphoviridae species were among the top-most important classifiers. AUC increased to 0.850 with solely antibiotic-exposed samples from 20 cachectic and 10 non-cachectic patients. This study is the first to suggest a link between cancer cachexia and intestinal bacteriophage richness. This could constitute a new basis for hypothesis-driven research in cancer cachexia diagnosis and treatment.

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Section: Discussionsupporting

confidence: 63%

“…Our work supports a relative high output for detection of bacteriophages in human gut by using shotgun metagenomes of stool samples without enrichment for viral-like particles (VLPs). This is important as shotgun metagenomes are much more readily available than VLP metagenomes 13,14 .…”

Section: Discussionmentioning

confidence: 99%

Lower abundance of human gut virus species is associated with cancer cachexia

Stang

Weilert

Illig

et al. 2022

Preprint

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“…The fecal samples of the donors for FVT were collected from a group of 11 normal-weight children aged 7-10 [6]. The samples were processed to isolate VLPs using the method previously described by our group [44]. Briefly, ∼250 mg of each fecal sample was resuspended in 40 mL of sterile buffer SM (100mM Sodium chloride, 10mM Magnesium sulfate, 50mM Tris-HCl, pH 7.5) and centrifuged at 4,700 x g for 30 min at room temperature and centrifuged at 4,700 x g for 30 min at room temperature to precipitate large debris.…”

Section: Methodsmentioning

confidence: 99%

“…The VLP isolation was conducted using the previously reported extraction protocol [6]. Following the previously reported protocol, the VLP numbers were determined using epifluorescence microscopy [44]. The DNA of VLPs was then extracted, following the manufacturer’s protocol for the QIAamp MinElute Virus Spin Kit (Cat.…”

Section: Methodsmentioning

confidence: 99%

Fecal Virome Transplantation (FVT) from healthy humans remodels the gut bacteriome and virome and reduces metabolic syndrome in mice

Cervantes-Echeverria,

Jimenez-Rico,

Manzo

et al. 2024

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The gut microbiome regulates host processes, including metabolism, and comprises viruses, archaea, fungi, protists, and bacteria. However, previous studies have primarily focused on the bacterial components of the microbiome. This study conducted cross-infection experiments by fecal virome transplantation (FVT) to examine the impact of virus-like particles (VLPs) from normal-weight individuals on bacterial and virome diversity and metabolic changes in mice. We found that FVT significantly restored glucose tolerance in mice, even though they were maintained on a high-fat diet (HFD). Shotgun metagenomics and 16s profiling confirmed that FVT shifted gut bacterial and viral richness and diversity, resulting in several differentially abundant bacteria and phages. The abundance of Akkermansia muciniphila decreased significantly after FVT, remained low until the end of the experiment (week 17), and correlated with a decreased glucose tolerance. On the other hand, there was a significant increase in Allobaculum and Coprococcus at weeks 10 and 17 post-FVT. The abundance of Allobaculum correlated with an increased glucose tolerance, indicating a beneficial hypoglycemic effect on the mice inoculated with VLPs. We also found an increased virome diversity at week 17, while richness was similar to the one before FVT. The study provides a proof of concept for the efficacy of FVT from human donors to mice, improving glucose tolerance by altering the gut microbiota and virome despite maintaining a HFD. Overall, our results suggest that trans-kingdom interactions between the virome and bacteriome from humans could be translated to mice as a study model.

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“…Recent advances in bioinformatics and experimental protocols for viral metagenomics showed us that the gut virome is mainly dominated by bacteriophages (Bikel et al, 2022, Shkoporov & Hill, 2019). Indeed, there are disease-specific changes in the gut virome in inflammatory bowel disease (Norman et al, 2015; Zuo et al, 2019), AIDS (Monaco et al, 2016), diabetes (Ma et al, 2018), malnutrition (Reyes et al, 2015), obesity, and obesity with metabolic syndrome (Bikel et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

A loss of crAssphage stability in the human gut virome is associated with obesity and metabolic syndrome

Cervantes-Echeverria

Gallardo-Becerra

Cornejo‐Granados

et al. 2022

Preprint

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Viral metagenomics studies of the human gut microbiota unravel differences in phage populations between healthy and disease, stimulating interest in the role that phages play in bacterial ecosystem regulation. CrAssphages are not only the most abundant viruses but also are a common component of the gut phageome across human populations. However, the role of crAssphages in obesity (O) and obesity with metabolic syndrome (OMS) remains largely unknown. Therefore, we explored the role that crAssphages have on both diseases in a children's cohort. We found decreased crAssphage abundance, prevalence, richness, and diversity in O and OMS compared to normal-weight (NW), suggesting a loss of crAssphages stability in the human phageome associated with the disease. Interestingly, when we analyzed the abundance of host crAssphages bacteria, we found that Bacteroidetes, Bacteroidia, and Bacteroidales were significantly decreased in O and OMS, suggesting a possible relation with the loss of crAssphages stability. Regarding crAssphage taxonomy, a significantly decreased abundance of the crAssphage Alpha subfamily and the Alpha_1 and Alpha_4 genus and a significant overabundance of the Delta_8 was found in OMS. A strong taxonomical signature of obesity is the over-abundance of Bacilli, which also were significantly increased in O and OMS. Notably, we found a significant negative correlation between crAssphages and Bacilli abundances, suggesting an association between the decreased abundance of crAssphage and the over-abundance of Bacilli in OMS. Furthermore, we found a loss of crAssphage stability in the human virome associated with the presence of obesity, having a more significant impact on obesity with metabolic syndrome, suggesting that these bacteriophages could play an essential role in inhibiting metabolic syndrome in obese individuals. Our results open a promising treatment for these diseases through fecal crAssphage transplantation.

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Protocol for the isolation, sequencing, and analysis of the gut phageome from human fecal samples

Cited by 6 publications

References 21 publications

Lower abundance of human gut virus species is associated with cancer cachexia

Lower abundance of human gut virus species is associated with cancer cachexia

Fecal Virome Transplantation (FVT) from healthy humans remodels the gut bacteriome and virome and reduces metabolic syndrome in mice

A loss of crAssphage stability in the human gut virome is associated with obesity and metabolic syndrome

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