Search

The first step in phage infection is the recognition of, and adsorption to, a receptor located on the host cell surface. This reversible host adsorption step is commonly followed by an irreversible event, which involves phage DNA delivery or release into the bacterial cytoplasm. The molecular components that trigger this latter event are unknown for most phages of Gram-positive bacteria. In the current study, we present a comparative genome analysis of three mutants of Lactococcus cremoris 3107, which are resistant to the P335 group phage TP901-1 due to mutations that affect TP901-1 DNA release. Through genetic complementation and phage infection assays, a predicted lactococcal three-component glycosylation system (TGS) was shown to be required for TP901-1 infection. Major cell wall saccharidic components were analysed, but no differences were found. However, heterologous gene expression experiments indicate that this TGS is involved in the glucosylation of a cell envelope-associated component that triggers TP901-1 DNA release. To date, a saccharide modification has not been implicated in the DNA delivery process of a Gram-positive infecting phage.

Ultraviolet C (UVC) radiation is a widely used technology for the disinfection of surfaces, air flows, water and other liquids. Although extensive research has been conducted on the UV tolerance of bacteriophages used as surrogates for waterborne viruses, limited information is available on phages relevant to food processing. Phages of dairy starters may reach high numbers in dairy facilities and cause fermentation failure with great economic losses for the dairy industry. Here, the UV tolerance of virulent phages, belonging to the 936-group (Skunavirus) of Lactococcus lactis subsp. diacetylactis F7/2, was assessed, employing both host infectivity loss and qPCR assays. A highly heat-tolerant phage (P680) and a less heat-tolerant phage (P008) were exposed to UV radiation at 265 nm (UVC), 285 nm (UVB) and 365 nm (UVA), respectively, in an aqueous suspension, using UV Light-Emitting-Diodes (LEDs) in a static set-up. UVC at 265 nm achieved the highest total inactivation, leading to a 4 log10 reduction of the phage titer at a UV dose of 327 and 164 mJ/cm2 for P680 and P008, respectively. UVB at 285 nm achieved similar inactivation levels, while UVA at 365 nm did not cause major reductions. Phages were also suspended in yoghurt serum of pH 5.5 and pH 7.0 and exposed to UVC radiation at 265 nm. The heat-tolerant phage P680 was more UV tolerant for all wavelengths, matrices and pH values tested. A higher aggregation degree together with less DNA damage was observed for both phages at pH 5.5, especially for phage P680, indicating a UV light-shielding effect. Interestingly, there were indications of some phage survivors exhibiting higher UV tolerance on re-exposure, pointing out a need for further investigation. Our results show that UV LEDs emitting at 265 nm and 285 nm are efficient in reducing the phage population significantly, but also underline that 936-type phages are relatively UV resistant. A further understanding of the main factors influenc

Eggerthella lenta is a common member of the human gut microbiome. We here describe the isolation and characterization of a putative virulent bacteriophage having E. lenta as host. The double-layer agar method for isolating phages was adapted to anaerobic conditions for isolating bacteriophage PMBT5 from sewage on a strictly anaerobic E. lenta strain of intestinal origin. For this, anaerobically grown E. lenta cells were concentrated by centrifugation and used for a 24 h phage enrichment step. Subsequently, this suspension was added to anaerobically prepared top (soft) agar in Hungate tubes and further used in the double-layer agar method. Based on morphological characteristics observed by transmission electron microscopy, phage PMBT5 could be assigned to the Siphoviridae phage family. It showed an isometric head with a flexible, noncontractile tail and a distinct single 45 nm tail fiber under the baseplate. Genome sequencing and assembly resulted in one contig of 30,930 bp and a mol% GC content of 51.3, consisting of 44 predicted protein-encoding genes. Phage-related proteins could be largely identified based on their amino acid sequence, and a comparison with metagenomes in the human virome database showed that the phage genome exhibits similarity to two distantly related phages.

In the present study, we describe the identification of potential citrate metabolism pathways for the lactic acid bacterium (LAB) Carnobacterium maltaromaticum. A phenotypic assay indicated that four of six C. maltaromaticum strains showed weak (Cm 6-1 and ATCC 35586) or even delayed (Cm 3-1 and Cm 5-1) citrate utilization activity. The remaining two strains, Cm 4-1 and Cm 1-2 gave negative results. Additional analysis showed no or very limited utilization of citrate in media containing 1% glucose and 22 or 30 mM citrate and inoculated with Cm 6-1 or ATCC 35586. Two potential pathways of citrate metabolism were identified by bioinformatics analyses in C. maltaromaticum including either oxaloacetate (pathway 1) or tricarboxylic compounds such as isocitrate and α-ketoglutarate (pathway 2) as intermediates. Genes encoding pathway 1 were present in two out of six strains while pathway 2 included genes present in all six strains. The two potential citrate metabolism pathways in C. maltaromaticum may potentially affect the sensory profiles of milk and soft cheeses subjected to growth with this species.

A bacterial strain, designated WCA-9-b2T, was isolated from the caecal content of an 18-week-old obese C57BL/6NTac male mouse. According to phenotypic analyses, the isolate was rod-shaped, strictly anaerobic, spore-forming, non-motile and Gram-stain-positive, under the conditions tested. Colonies were irregular and non-pigmented. Analysis of the 16S rRNA gene sequence indicated that the isolate belonged to the order Clostridiales with Dorea longicatena ATCC 27755T (94.9 % sequence identity), Ruminococcus gnavus ATCC 29149T (94.8%) and Clostridium scindens ATCC 35704T (94.3%) being the closest relatives. Whole genome sequencing showed an average nucleotide identity <74.23 %, average amino acid identity <64.52–74.67 % and percentage of conserved proteins values <50 % against the nine closest relatives (D. longicatena, Ruminococcus gnavus, C. scindens, Dorea formicigenerans, Ruminococcus lactaris, Clostridium hylemonae, Merdimonas faecis, Faecalicatena contorta and Faecalicatena fissicatena). The genome-based G+C content of genomic DNA was 44.4 mol%. The major cellular fatty acids were C16: 0 (24.5%), C18: 1 cis9 (19.8 %), C16: 0 DMA (11.7%), C18: 0 (8.4%) and C14: 0 (6.6%). Respiratory quinones were not detected. The predominant metabolic end products of glucose fermentation were acetate and succinate. Production of CO2 and H2 were detected. Based on these data, we propose that strain WCA-9-b2T represents a novel species within a novel genus, for which the name Sporofaciens musculi gen. nov., sp. nov. is proposed. The type strain is WCA-9-b2T (=DSM 106039T=CECT 30156T).

Lactococcus lactis phage P335 is a virulent type phage for the species that bears its name and belongs to the Siphoviridae family. Morphologically, P335 resembled the L. lactis phages TP901-1 and Tuc2009, except for a shorter tail and a different collar/whisker structure. Its 33,613-bp double-stranded DNA genome had 50 open reading frames. Putative functions were assigned to 29 of them. Unlike other sequenced genomes from lactococcal phages belonging to this species, P335 did not have a lysogeny module. However, it did carry a dUTPase gene, the most conserved gene among this phage species. Comparative genomic analyses revealed a high level of identity between the morphogenesis modules of the phages P335, ul36, TP901-1, and Tuc2009 and two putative prophages of L. lactis SK11. Differences were noted in genes coding for receptor-binding proteins, in agreement with their distinct host ranges. Sixteen structural proteins of phage P335 were identified by liquid chromatography-tandem mass spectrometry. A 2.8-kb insertion was recognized between the putative genes coding for the activator of late transcription (Alt) and the small terminase subunit (TerS). Four genes within this region were autonomously late transcribed and possibly under the control of Alt. Three of the four deduced proteins had similarities with proteins from Streptococcus pyogenes prophages, suggesting that P335 acquired this module from another phage genome. The genetic diversity of the P335 species indicates that they are exceptional models for studying the modular theory of phage evolution.

Gut microbiome (GM) composition and function are linked to human health and disease, and routes for manipulating the GM have become an area of intense research. Due to its high treatment efficacy, the use of fecal microbiota transplantation (FMT) is generally accepted as a promising experimental treatment for patients suffering from GM imbalances (dysbiosis), e.g. caused by recurrent Clostridioides difficile infections (rCDI). Mounting evidence suggests that bacteriophages (phages) play a key role in successful FMT treatment by restoring the dysbiotic bacterial GM. As a refinement to FMT, removing the bacterial component of donor feces by sterile filtration, also referred to as fecal virome transplantation (FVT), decreases the risk of invasive infections caused by bacteria. However, eukaryotic viruses and prophage-encoded virulence factors remain a safety issue. Recent in vivo studies show how cascading effects are initiated when phage communities are transferred to the gut by e.g. FVT, which leads to changes in the GM composition, host metabolome, and improve host health such as alleviating symptoms of obesity and type-2-diabetes (T2D). In this review, we discuss the promises and limitations of FVT along with the perspectives of using FVT to treat various diseases associated with GM dysbiosis.

Objective: Development of obesity and type 2 diabetes (T2D) are associated with gut microbiota (GM) changes. The gut viral community is predominated by bacteriophages (phages), which are viruses that attack bacteria in a host-specific manner. The antagonistic behaviour of phages has the potential to alter the GM. As a proof-of-concept, we demonstrate the efficacy of faecal virome transplantation (FVT) from lean donors for shifting the phenotype of obese mice into closer resemblance of lean mice. Design: The FVT consisted of viromes with distinct profiles extracted from the caecal content of mice from different vendors that were fed a low-fat (LF) diet for 14 weeks. Male C57BL/6NTac mice were divided into five groups: LF (as diet control), high-fat (HF) diet, HF+ampicillin (Amp), HF+Amp+FVT and HF+FVT. At weeks 6 and 7 of the study, the HF+FVT and HF+Amp+FVT mice were treated with FVT by oral gavage. The Amp groups were treated with Amp 24 hours prior to first FVT treatment. Results: Six weeks after first FVT, the HF+FVT mice showed a significant decrease in weight gain compared with the HF group. Further, glucose tolerance was comparable between the LF and HF+FVT mice, while the other HF groups all had impaired glucose tolerance. These observations were supported by significant shifts in GM composition, blood plasma metabolome and expression levels of genes associated with obesity and T2D development. Conclusions: Transfer of caecal viral communities from mice with a lean phenotype into mice with an obese phenotype led to reduced weight gain and normalised blood glucose parameters relative to lean mice. We hypothesise that this effect is mediated via FVT-induced GM changes.

Often physiological studies using mice from one vendor show different outcome when being reproduced using mice from another vendor. These divergent phenotypes between similar mouse strains from different vendors have been assigned to differences in the gut microbiome. During recent years, evidence has mounted that the gut viral community plays a key role in shaping the gut microbiome and may thus also influence mouse phenotype. However, to date inter-vendor variation in the murine gut virome has not been studied. Using a metavirome approach, combined with 16S rRNA gene sequencing, we here compare the composition of the viral and bacterial gut community of C57BL/6N mice from three different vendors exposed to either a chow-based low-fat diet or high-fat diet. Interestingly, both the bacterial and the viral component of the gut community differed significantly between vendors. The different diets also strongly influenced both the viral and bacterial gut community, but surprisingly the effect of vendor exceeded the effect of diet. In conclusion, the vendor effect is substantial not only on the gut bacterial community but also strongly influences viral community composition. Given the effect of GM on mice phenotype, this is essential to consider for increasing reproducibility of mouse studies.