Bacillus safensis APC 4099, isolated from bees' gut, has been identified as a promising candidate for food biopreservation. Antimicrobial activity screening revealed a broad-spectrum inhibition potential, ranging from gram-positive pathogenic bacteria to fungi responsible for food spoilage. Genomic analysis identified biosynthetic gene clusters coding for several antimicrobial peptides and secondary metabolites. Specifically, a novel, anionic, 6 kDa circular bacteriocin, named safencin E, was detected, showing 52.5% similarity to butyrivibriocin AR10. Additionally, gene clusters coding for the biosynthesis of bacteriocins such as pumilarin and plantazolicin and biosynthetic pathways for secondary metabolites, including pumilacidin A, bacilysin, and bacillibactin, were identified. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis detected molecular masses correlating to safencin E, plantazolicin, pumilarin, and pumilacidin A from the cell-free supernatant, cell extracts, or both. Overall, the broad-spectrum antimicrobial activity of B. safensis APC 4099 indicates that this strain is a promising candidate for the biological control of food ecosystems and thus has the potential to enhance food safety. The present article highlights the importance of the strain Bacillus safensis APC 4099 as a potential biocontrol agent. The strain possesses biosynthetic gene clusters coding for various antimicrobial peptides and secondary metabolites, including a novel circular bacteriocin, safencin E, and the bacteriocins pumilarin and plantazolicin. This diversity in the production of antimicrobial peptides renders the producer with broad-spectrum antimicrobial activity, ranging from gram-positive pathogenic and spoilage bacteria to spoilage molds. Considering that 1.3 billion tons of food appropriate for human consumption is lost or wasted annually, identifying strains or novel antimicrobial peptides capable of biopreservation is highly relevant. This strain and its bioactive compounds offer a solution to this global problem as biocontrol agents for food ecosystems against spoilage and pathogenic microbes.
Abstract The global rise of antibiotic-resistant bacteria, particularly among Streptococcus species, poses an escalating public health threat. Traditional antibiotic development has proven inadequate, making innovative approaches such as bacteriophage-based therapies promising alternatives. A deep understanding of phage biology at the genomic level is essential for advancing therapeutic applications. Here, we analyzed 709 Streptococcus phage genomes to bridge gaps in genomic diversity and propose revisions to Streptococcus phage taxonomy. The phage genomes were clustered based on shared proteins, resulting in 66 clusters and 35 singletons with significant variation in genome characteristics. Through proteome phylogeny, average nucleotide identity, and inter-cluster core genes, we propose 21 new family-level classifications and 296 genus-level subclusters, providing an updated framework for Streptococcus phage taxonomy. Further analysis revealed diverse domain architectures in Streptococcus phage endolysins, including previously unreported structures. Specific domains were associated with distinct streptococcal hosts, suggesting adaptive evolution. We also observed variation in endolysin gene organization, with purifying selection acting on most sites, though some were subject to diversifying selection. Additionally, 182 novel endolysin-derived antimicrobial peptides (AMPs) were identified, some exhibiting antifungal, antiviral, cell-penetrating and non-toxic properties. Molecular dynamics and docking simulations demonstrated high stability and strong binding affinity of peptides EP-39 and EP-121 to the Streptococcus pneumoniae virulence factor autolysin. This is the first comprehensive comparative study of Streptococcus phage genomes, providing critical insights into phage diversity and taxonomy. It also highlights the therapeutic potential of endolysin-derived AMPs against multidrug-resistant Streptococcus strains. Further experimental validation is required to assess their clinical potential.
Ruminococcus gnavus is a prevalent gut microbe reported to occur in higher abundance among individuals with inflammatory bowel disease (IBD). This study reports the isolation and characterization of six bacteriophages (phages) isolated from human fecal material and environmental samples that infect this species. Isolated phages have a siphovirus morphology, with genomes ranging between 36.5 and 37.8 kbp. Genome analysis indicates that the phages have a temperate lifestyle, which was confirmed by their ability to form lysogens on their host bacterial species. In contrast to the finding that phages lyse their host in liquid medium, results from a mouse trial indicate these phages can co-exist with the host bacterium in the gut without causing a significant reduction of R. gnavus. The bacterial counts in the feces of phage-treated mice did not significantly differ in the presence of phage. Furthermore, analysis of publicly available gut virome sequence data indicates a high abundance of these phages among individuals suffering from IBD. This work provides the first insight into how phages interact with R. gnavus in the human gut microbiome.
Losses in crop yields due to disease need to be reduced in order to meet increasing global food demands associated with growth in the human population. There is a well recognised need to develop new environmentally-friendly control strategies to combat bacterial crop disease. Current control measures involving the use of traditional chemicals or antibiotics are losing their efficacy due to the natural development of bacterial resistance to these agents. In addition, there is an increasing awareness that their use is environmentally unfriendly. Bacteriophages, the viruses of bacteria, have received increased research interest in recent years as a realistic environmentally friendly means of controlling bacterial diseases. Their use presents a viable control measure for a number of destructive bacterial crop diseases, with some phage-based products already becoming available on the market. Phage biocontrol possesses advantages over chemical controls in that tailor-made phage cocktails can be adapted to target specific disease-causing bacteria. Unlike chemical control measures, phage mixtures can be easily adapted for bacterial resistance which may develop over time. In this review, we will examine the progress and challenges for phage-based disease biocontrol in food crops.
Pectobacterium atrosepticum is a phytopathogen of economic importance as it is the causative agent of potato blackleg and soft rot. Here we describe the Pectobacterium phage vB_PatP_CB5 (abbreviated as CB5), which specifically infects the bacterium. The bacteriophage is characterized in detail and TEM micrographs indicate that it belongs to the Podoviridae family. CB5 shares significant pairwise nucleotide identity (≥80%) with P. atrosepticum phages φM1, Peat1, and PP90 and also shares common genome organization. Phylograms constructed using conserved proteins and whole-genome comparison-based amino acid sequences show that these phages form a distinct clade within the Autographivirinae. They also possess conserved RNA polymerase recognition and specificity loop sequences. Their lysis cassette resembles that of KP34virus, containing in sequential order a U-spanin, a holin, and a signal⁻arrest⁻release (SAR) endolysin. However, they share low pairwise nucleotide identity with the type phage of the KP34virus genus, Klebsiella phage KP34. In addition, phage KP34 does not possess several conserved proteins associated with these P. atrosepticum phages. As such, we propose the allocation of phages CB5, Peat1, φM1, and PP90 to a separate new genus designated Phimunavirus.
Pectobacterium atrosepticum is an economically important phytopathogen that is responsible for potato blackleg and soft rot, and for which current control strategies are limited. In this study, stem samples of potato crops exhibiting blackleg were taken from three farms in Co. Cork, Ireland, and they were found to be infected with P. atrosepticum. Three closely related bacteriophages (phages) that are specific to this phytopathogen were isolated and characterized, namely vB_PatP_CB1, vB_PatP_CB3, and vB_PatP_CB4 (abbreviated as CB1, CB3, and CB4). Both CB1 and CB3 were determined to infect 12 strains and CB4 10 strains of the 19 strains of P. atrosepticum tested. Morphology, latent periods, burst sizes, and their stability at various temperatures and pHs were also examined. Genome sequencing of the three phages revealed that they shared a minimum nucleotide identity of 93% with each other. Their genomes exhibited an Enquartavirinae genome organization, possessing several conserved proteins that were associated with phages of this group, like the type species Escherichia virus N4. Tandem electrospray ionization-mass spectrometry (ESI-MS/MS) allowed for the identification of ten structural proteins that form the virion of CB1, six that are conserved in phage N4. Biocontrol experiments demonstrated that the phages suppress soft rot formation upon co-inoculation with P. atrosepticum on whole tubers. The results of this study indicate that CB1 related phages could be good candidates for phage-based control.
Escherichia coli and Enterococcus faecalis have been implicated as important players in human gut health that have been associated with the onset of inflammatory bowel disease (IBD). Bacteriophage (phage) therapy has been used for decades to target pathogens as an alternative to antibiotics, but the ability of phage to shape complex bacterial consortia in the lower gastrointestinal tract is not clearly understood. We administered a cocktail of six phages (either viable or heat-inactivated) targeting pro-inflammatory Escherichia coli LF82 and Enterococcus faecalis OG1RF as members of a defined community in both a continuous fermenter and a murine colitis model. The two target strains were members of a six species simplified human microbiome consortium (SIHUMI-6). In a 72-h continuous fermentation, the phage cocktail caused a 1.1 and 1.5 log (log 10 genome copies/mL) reduction in E. faecalis and E. coli numbers, respectively. This interaction was accompanied by changes in the numbers of other SIHUMI-6 members, with an increase of Lactiplantibacillus plantarum (1.7 log) and Faecalibacterium prausnitzii (1.8 log). However, in germ-free mice colonized by the same bacterial consortium, the same phage cocktail administered twice a week over nine weeks did not cause a significant reduction of the target strains. Mice treated with active or inactive phage had similar levels of pro-inflammatory cytokines (IFN-y/IL12p40) in unstimulated colorectal colonic strip cultures. However, histology scores of the murine lower GIT (cecum and distal colon) were lower in the viable phage-treated mice, suggesting that the phage cocktail did influence the functionality of the SIHUMI-6 consortium. For this study, we conclude that the observed potential of phages to reduce host populations in in vitro models did not translate to a similar outcome in an in vivo setting, with this effect likely brought about by the reduction of phage numbers during transit of the mouse GIT.
ABSTRACT Escherichia coli is a commensal inhabitant of the mammalian gut microbiota, frequently associated with various gastrointestinal diseases. There is increasing interest in comprehending the variety of bacteriophages (phages) that target this bacterium, as such insights could pave the way for their potential use in therapeutic applications. Here, we report the isolation and characterization of four newly identified E. coli infecting tailed phages (W70, A7-1, A5-4, and A73) that were found to constitute a novel genus, Septuagintavirus , within the subfamily Vequintavirinae . Genomes of these phages ranged from 137 kbp to 145 kbp, with a GC content of 41 mol%. They possess a maximum nucleotide similarity of 30% with phages of the closest phylogenetic genus, Certrevirus , while displaying limited homology to other genera of the Vequintavirinae family. Host range analysis showed that these phages have limited activity against a panel of E. coli strains, infecting 6 out of 16 tested isolates, regardless of their phylotype. Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) was performed on the virion of phage W70, allowing the identification of 28 structural proteins, 19 of which were shared with phages of other genera of Vequintavirinae family. The greatest diversity was identified with proteins forming tail fiber structures, likely indicating the adaptation of virions of each phage genus of this subfamily for the recognition of their target receptor on host cells. The findings of this study provide greater insights into the phages of the subfamily Vequintavirinae , contributing to the pool of knowledge currently known about these phages. IMPORTANCE Escherichia coli is a well-known bacterium that inhabits diverse ecological niches, including the mammalian gut microbiota. Certain strains are associated with gastrointestinal diseases, and there is a growing interest in using bacteriophages, viruses that infect bacteria, to combat bacterial infections. Here, we describe the isolation and characterization of four novel E. coli bacteriophages that constitute a new genus, Septuagintavirus , within the subfamily Vequintavirinae . We conducted mass spectrometry on virions of a representative phage of this novel clade and compared it to other phages within the subfamily. Our analysis shows that virion structure is highly conserved among all phages, except for proteins related to tail fiber structures implicated in the host range. These findings provide greater insights into the phages of the subfamily Vequintavirinae , contributing to the existing pool of knowledge about these phages.
Streptococcus pneumoniae is highly pathogenic and causes several mucosal and invasive infections. Due to the rising number of multidrug-resistant (MDR) strains of S. pneumoniae, new antimicrobials with alternative mechanisms of action are urgently needed. In this study, we identified two new Streptococcal phages from the oral microbiome, 23TH and SA01. Their lysins, 23TH_48 and SA01_53, were recombinantly expressed, characterized and tested for their lethality. SA01_53 was found to only lyse its host strain of S. anginosus, while 23TH_48 was found to possess a broader lytic activity beyond its host strain of S. infantis, with several S. pneumoniae isolates sensitive to its lytic activity. 23TH_48 at a concentration of five activity units per mL (U/mL) was found to reduce cell counts of S. pneumoniae DSM 24048 by 4 log10 colony forming units per mL (CFU/mL) within 1 h and effectively prevented and destroyed biofilms of S. pneumoniae R6 at concentrations of 228.8 ng/µL and 14.3 ng/µL, respectively. Given its high lytic activity, 23TH_48 could prove to be a promising candidate to help combat pneumococcal infections.
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