Search Results (54)

Phage display has advanced the discovery of peptides that selectively bind to a wide variety of cell surface molecules, offering new modalities to modulate disease-related protein–protein interactions (PPIs). These cell-binding peptides occupy a unique pharmaceutical space between small molecules and large biologics, and their growing popularity has opened up new avenues for targeting cell surface proteins that were previously considered undruggable. This work provides an overview of methods for identifying cell-selective peptides using phage display combinatorial libraries, covering in vitro, ex vivo, and in vivo biopanning approaches. It addresses key considerations in library design, including the peptide conformation (linear vs. cyclic) and length, and highlights examples of clinically approved peptides developed through phage display. It also discusses the on-phage chemical cyclization of peptides to overcome the limitations of genetically encoded disulfide bridges and emphasizes advances in combining next-generation sequencing (NGS) with phage display to improve peptide selection and analysis workflows. Furthermore, due to the often suboptimal binding affinity of peptides identified in phage display selections, this article discusses affinity maturation techniques, including random mutagenesis and rational design through structure–activity relationship (SAR) studies to optimize initial peptide candidates. By integrating these developments, this review outlines practical strategies and future directions for harnessing phage display in targeting challenging cell surface proteins. Full article

HLB is a severe and devastating disease affecting citrus plants, for which no cure is currently available. The pathogen CLas is an unculturable, phloem-limited α-proteobacterium associated with HLB. Phages, known for their ability to directly lyse pathogenic bacteria, have been widely recognized as effective control agents in both medical and agricultural fields. In this study, we identified 191 prophages across 48 Liberibacter genomes using comparative genomics and phylogenetic methods. These prophages were classified into 17 distinct types, with 13 novel types, named NF1 to NF13. Diversity analysis revealed that the number of prophages per genome ranged from one to eight, with an average of four. Annotation showed that the genome sizes of NF1–NF13 ranged from 10,160 bp to 72,736 bp, with an average size of 27,616 bp, containing between 7 and 66 genes (average: 27 genes). Further functional enrichment of the sequences within 5 kb upstream and downstream of the integration site identified six main aspects, involving four core biological processes: genome structure modification, gene expression regulation, stress response activation, and metabolic pathway modulation. It is speculated that the insertion of the prophage significantly impacts the host. Six virulence factors, including carB, clpC, flhA, algW, rffG, and IlpA, were identified in the prophages. Interestingly, prophages containing carB or clpC are predominantly found in CLas, which may contribute to the global colonization of CLas. Notably, among the 35 identified Type 4 prophages, 19 were found to be active. Type 4 is the only active prophage type, making it a promising candidate for developing phage therapy against CLas. This study is significant for a deeper understanding of the diversity of Liberibacter and the interactions between prophages and their hosts. Full article

Zearalenone (ZEN), a toxic estrogenic mycotoxin in cereals, threatens human and animal health through reproductive, immune, and cytotoxic effects, necessitating sensitive detection methods. While nanobodies offer advantages over conventional antibodies for on-site ZEN detection, their application remains unexplored. This study aimed to develop an anti-ZEN nanobody derived from an anti-ZEN phage display nanobody library. An alpaca was immunized with a ZEN-bovine serum albumin (ZEN-BSA) antigen, achieving peak serum antibody titers (1:25,600) following four immunizations. A high-capacity phage display nanobody library (1.0 × 10¹¹ plaque-forming units/mL) was constructed. Following four rounds of biopanning, an enrichment factor of 479 was achieved. Phage ELISA screening identified six phage display nanobodies with specific ZEN-binding activity, and multiple sequence alignment revealed four unique nanobody sequences. The selected phage display nanobody, designated phage-V44, was expressed and purified, and its presence was validated by SDS-PAGE and western blotting, which detected a single approximately 17 kDa band consistent with the expected nanobody size. We established a working curve for an indirect competitive enzyme-linked immunoassay (ELISA) for ZEN, which showed an IC50 value of 7.55 ng/mL. The specificity and affinity of the V44 were also verified. Collectively, the study successfully constructed an anti-ZEN phage display nanobody library, screened four specific ZEN-binding phage display nanobodies, and prepared the anti-ZEN nanobody V44. Thereby establishing a foundation for the nanobody’s future integration into rapid on-site detection methods for ZEN in both animal feed and human food products. Full article

Metabolic disorders, including type 2 diabetes mellitus (T2DM), obesity, and metabolic syndrome, are systemic conditions that profoundly impact the skin microbiota, a dynamic community of bacteria, fungi, viruses, and mites essential for cutaneous health. Dysbiosis caused by metabolic dysfunction contributes to skin barrier disruption, immune dysregulation, and increased susceptibility to inflammatory skin diseases, including psoriasis, atopic dermatitis, and acne. For instance, hyperglycemia in T2DM leads to the formation of advanced glycation end products (AGEs), which bind to the receptor for AGEs (RAGE) on keratinocytes and immune cells, promoting oxidative stress and inflammation while facilitating Staphylococcus aureus colonization in atopic dermatitis. Similarly, obesity-induced dysregulation of sebaceous lipid composition increases saturated fatty acids, favoring pathogenic strains of Cutibacterium acnes, which produce inflammatory metabolites that exacerbate acne. Advances in metabolomics and microbiome sequencing have unveiled critical biomarkers, such as short-chain fatty acids and microbial signatures, predictive of therapeutic outcomes. For example, elevated butyrate levels in psoriasis have been associated with reduced Th17-mediated inflammation, while the presence of specific Lactobacillus strains has shown potential to modulate immune tolerance in atopic dermatitis. Furthermore, machine learning models are increasingly used to integrate multi-omics data, enabling personalized interventions. Emerging therapies, such as probiotics and postbiotics, aim to restore microbial diversity, while phage therapy selectively targets pathogenic bacteria like Staphylococcus aureus without disrupting beneficial flora. Clinical trials have demonstrated significant reductions in inflammatory lesions and improved quality-of-life metrics in patients receiving these microbiota-targeted treatments. This review synthesizes current evidence on the bidirectional interplay between metabolic disorders and skin microbiota, highlighting therapeutic implications and future directions. By addressing systemic metabolic dysfunction and microbiota-mediated pathways, precision strategies are paving the way for improved patient outcomes in dermatologic care. Full article

Leukocidins of Staphylococcus (S.) aureus are bicomponent toxins that form polymeric pores in host leukocyte membranes, leading to cell death and/or triggering apoptosis. Some of these toxin genes are located on prophages and are associated with specific hosts. The genes lukP/Q have been described from equine S. aureus isolates. We examined the genomes, including the lukP/Q prophages, of S. aureus strains belonging to clonal complexes CC1, CC350, CC816, and CC8115. In addition to sequencing, phages were characterised by mitomycin C induction and transmission electron microscopy (TEM). All lukP/Q prophages integrated into the lip2=geh gene, and all included also the gene scn-eq encoding an equine staphylococcal complement inhibitor. The lukP/Q prophages clustered, based on gene content and allelic variants, into three groups. One was found in CC1 and CC97 sequences; one was present mainly in CC350 but also in other lineages (CC1, CC97, CC133, CC398); and a third one was exclusively observed in CC816 and CC8115. Prophages of the latter group additionally included a rare enterotoxin A allele (sea_320E). Moreover, a prophage from a CC522 goat isolate was found to harbour lukP. Its lukF component could be regarded as chimaera comprising parts of lukQ and of lukF-P83. A putative kinase gene of 1095 basepairs was found to be associated with equine strains of S. aureus. It was also localised on prophages. However, these prophages were different from the ones that carried lukP/Q, and three different integration sites of kinase-carrying phages were identified. These observations confirmed the presence of prophage-located important virulence-associated genes in equine S. aureus and that certain prophages might determine the host specificity of the staphylococcal strains they reside in. Full article

Background/Objectives: This study explores the genome sequencing data from the infection of Pseudomonas fluorescens UFV 041 by the bacteriophage Pijolavirus UFJF_PfSW6, aiming to identify and characterize prophages induced in the host bacterium during the infection. Methods: Scaffolds from sequencing data were analyzed, and reads were mapped to identify potential prophages using phage-to-host coverage metrics. The putative prophage scaffold was annotated, taxonomically classified, and its integration in the host bacterium was verified by PCR amplification of two target genes. We also tested whether mitomycin treatment could induce the prophage to enter the lytic cycle. Results: The prophage UFJF_PfPro was identified with a high phage-to-host coverage ratio. Its genome is 32,700 bp in length, containing 42 genes, 3 terminators, and 11 promoters, with 98.84% completeness. PCR confirmed its integration into P. fluorescens UFV 041, but mitomycin treatment did not induce the lytic cycle. The UFJF_PfPro genome shares 38.60% similarity with the closest lytic phages in the Phitrevirus genus, below genus and species assignment thresholds. A viral proteomic tree clustered UFJF_PfPro with Phitrevirus in a clade representing the Peduoviridae family. Conclusions: The UFJF_PfPro is a prophage integrated into the P. fluorescens UFV 041 genome, but we were unable to induce it to enter the lytic cycle using mitomycin treatment. The genome of UFJF_PfPro encodes all structural proteins typical of the Caudoviricetes class and shares low genomic similarity with species of the genus Phitrevirus, suggesting that UFJF_PfPro represents a new genus and species within the Peduoviridae family. Full article

Salmonella is one of the main foodborne pathogens. Irrational antibiotic management has led to an increase in the incidence of multidrug-resistant strains. Bacteriophages may be an alternative method of food biopreservation and contribute to reducing the number of food poisonings requiring pharmacotherapy. This study aimed to isolate a bacteriophage (phage) targeting indigenous multidrug-resistant (MDR) Salmonella strains, followed by their biological, morphological, and genomic characterization. In this study we isolated Salmonella phage KKP_3822, targeting MDR Salmonella Manchester strain KKP 1213. Salmonella phage KKP_3822 retained high activity in the temperature range from −20 °C to 40 °C and active acidity from pH 3 to 11. Temperatures of 70 °C and 80 °C and extreme pH values (2 and 12) significantly reduced the phage titer. Its activity decreased proportionally to the time of UV exposure. Genome analysis (linear dsDNA with a length of 114,843 bp) revealed the presence of 27 tRNA genes. Proteins encoded by the vB_Sen-IAFB3822 phage were divided into functional modules related to (i) phage structure/assembly, (ii) DNA replication/modification/regulation, (iii) phage lysis, and (iv) DNA packaging into the capsid. No genes associated with antibiotic resistance or integration into the host genome, markers of temperate bacteriophages, were annotated in the Salmonella phage KKP_3822 genome. Based on morphological features and whole-genome sequence analysis, the newly isolated Salmonella phage KKP_3822 shows the greatest similarity to representatives of tailed phages from the Caudoviricetes class, Demerecviridae family, and Epseptimavirus genus. Genome analysis confirmed the virulent nature of the Salmonella phage KKP_3822, making it a potential candidate for food biocontrol. Full article

Erysipelas is a significant problem in the waterfowl farming in Poland, and information on the characteristics of the Erysipelothrix rhusiopathiae strains causing this disease is limited. In this study, we determined the serotypes, antimicrobial susceptibility, and potential mechanisms of resistance gene transfer in E. rhusiopathiae isolates (n = 60) from domestic geese and ducks. We also developed a multiplex PCR for the detection of resistance genes. The antimicrobial susceptibility of the isolates was assessed using the broth microdilution method. Resistance genes, integrative conjugative element (ICE)-specific genes, phage-specific genes, and serotype determinants were detected by PCR. Multilocus sequence typing (MLST) was performed for selected resistant strains. The comparative analyses included 260 E. rhusiopathiae strains whose whole genome sequences (WGSs) are publicly available. E. rhusiopathiae isolates represented 7 serotypes, among which serotypes 5 (38.3%) and 1b (28.3%) were the most common. All strains were susceptible to β-lactams, and the vast majority of them were resistant to tetracycline (85%) and enrofloxacin (80%). The percentages of isolates resistant to other antimicrobials used ranged from 3.3% to 16.7%. Ten isolates (16.7%) were found to be multidrug resistant (MDR). The genotypic resistance profiles of the E. rhusiopathiae strains corresponded to their phenotypic resistance, and the amplification patterns obtained using the 10-plex PCR developed in this study were fully consistent with the results of single PCRs. The most prevalent resistance gene was tetM. In enrofloxacin-resistant strains, nonsynonymous mutations in the gyrA and parC genes were identified. The presence of ICE-specific genes was confirmed in resistant strains, and in MDR isolates of serotype 8 that represented sequence type (ST) 113, prophage DNA (Javan630-like) linked to the lsaE gene was additionally detected. The results indicate that β-lactam antibiotics should be the first choice for the treatment of waterfowl erysipelas in Poland. ICEs, including a transposon from the Tn916/Tn1545 family, and bacteriophages are most likely responsible for the transfer of resistance genes in E. rhusiopathiae. Full article

Advancements in genomics and machine learning have significantly enhanced the study of Salmonella epidemiology. Whole-genome sequencing has revolutionized bacterial genomics, allowing for detailed analysis of genetic variation and aiding in outbreak investigations and source tracking. Short-read sequencing technologies, such as those provided by Illumina, have been instrumental in generating draft genomes that facilitate serotyping and the detection of antimicrobial resistance. Long-read sequencing technologies, including those from Pacific Biosciences and Oxford Nanopore Technologies, offer the potential for more complete genome assemblies and better insights into genetic diversity. In addition to these sequencing approaches, machine learning techniques like decision trees and random forests provide powerful tools for pattern recognition and predictive modeling. Importantly, the study of bacteriophages, which interact with Salmonella, offers additional layers of understanding. Phages can impact Salmonella population dynamics and evolution, and their integration into Salmonella genomics research holds promise for novel insights into pathogen control and epidemiology. This review revisits the history of Salmonella and its pathogenesis and highlights the integration of these modern methodologies in advancing our understanding of Salmonella. Full article

The diversity of phage-related sequences (PRSs) and their site-specific integration into the genomes of nonpathogenic, agriculturally valuable, nitrogen-fixing root nodule bacteria, such as Sinorhizobium meliloti, were evaluated in this study. A total of 314 PRSs, ranging in size from 3.24 kb to 88.98 kb, were identified in the genomes of 27 S. meliloti strains. The amount of genetic information foreign to S. meliloti accumulated in all identified PRSs was 6.30 Mb. However, more than 53% of this information was contained in prophages (Phs) and genomic islands (GIs) integrated into genes encoding tRNAs (tRNA genes) located on the chromosomes of the rhizobial strains studied. It was found that phiLM21-like Phs were predominantly abundant in the genomes of S. meliloti strains of distant geographical origin, whereas RR1-A- and 16-3-like Phs were much less common. In addition, GIs predominantly contained fragments of phages infecting bacteria of distant taxa, while rhizobiophage-like sequences were unique. A site-specific integration analysis revealed that not all tRNA genes in S. meliloti are integration sites, but among those in which integration occurred, there were “hot spots” of integration into which either Phs or GIs were predominantly inserted. For the first time, it is shown that at these integration “hot spots”, not only is the homology of attP and attB strictly preserved, but integrases in PRSs similar to those of phages infecting the Proteobacteria genera Azospirillum or Pseudomonas are also present. The data presented greatly expand the understanding of the fate of phage-related sequences in host bacterial genomes and also raise new questions about the role of phages in bacterial–phage coevolution. Full article

CTXΦ is a lysogenic filamentous phage that carries the genes encoding cholera toxin (ctxAB), the main virulence factor of Vibrio cholerae. The toxigenic conversion of environmental V. cholerae strains through CTXΦ lysogenic infection is crucial for the emergence of new pathogenic clones. A special allelic form of CTXΦ, called pre-CTXΦ, is a precursor of CTXΦ and without ctxAB. Different members of the pre-CTXΦ and CTXΦ families are distinguished by the sequence of the transcriptional repressor-coding gene rstR. Multiple rstR alleles can coexist within a single strain, demonstrating the diverse structure and complex genomic integration patterns of CTXΦ/pre-CTXΦ prophage on the chromosome. Exploration of the diversity and co-integration patterns of CTXΦ/pre-CTXΦ prophages in V. cholerae can help to understand the evolution of this phage family. In this study, 21 V. cholerae strains, which were shown to carry the CTXΦ/pre-CTXΦ prophages as opposed to typical CTX^ETΦ-RS1 structure, were selected from approximately 1000 strains with diverse genomes. We identified two CTXΦ members and six pre-CTXΦ members with distinct rstR alleles, revealing complex chromosomal DNA integration patterns and arrangements of different prophages in these strains. Promoter activity assays showed that the transcriptional repressor RstR protected against CTXΦ superinfection by preventing the replication and integration of CTXΦ/pre-CTXΦ phages containing the same rstR allele, supporting the co-integration of the diverse CTXΦ/pre-CTXΦ members observed. The numbers and types of prophages and their co-integration arrangements in serogroup O139 strains were more complex than those in serogroup O1 strains. Also, these CTXΦ/pre-CTXΦ members were shown to present the bloom period of the CTXΦ/pre-CTXΦ family during wave 2 of the seventh cholera pandemic. Together, these analyses deepen our comprehension of the genetic variation of CTXΦ and pre-CTXΦ and provide insights into the evolution of the CTXΦ/pre-CTXΦ family in the seventh cholera pandemic. Full article

The last thirty years have seen a meteoric rise in the number of sequenced bacteriophage genomes, spurred on by both the rise and success of groups working to isolate and characterize phages, and the rapid and significant technological improvements and reduced costs associated with sequencing their genomes. Over the course of these decades, the tools used to glean evolutionary insights from these sequences have grown more complex and sophisticated, and we describe here the suite of computational and bioinformatic tools used extensively by the integrated research–education communities such as SEA-PHAGES and PHIRE, which are jointly responsible for 25% of all complete phage genomes in the RefSeq database. These tools are used to integrate and analyze phage genome data from different sources, for identification and precise extraction of prophages from bacterial genomes, computing “phamilies” of related genes, and displaying the complex nucleotide and amino acid level mosaicism of these genomes. While over 50,000 SEA-PHAGES students have primarily benefitted from these tools, they are freely available for the phage community at large. Full article

Goose erysipelas is a serious problem in waterfowl breeding in Poland. However, knowledge of the characteristics of Erysipelothrix rhusiopathiae strains causing this disease is limited. In this study, the antimicrobial susceptibility and serotypes of four E. rhusiopathiae strains from domestic geese were determined, and their whole-genome sequences (WGSs) were analyzed to detect resistance genes, integrative and conjugative elements (ICEs), and prophage DNA. Sequence type and the presence of resistance genes and transposons were compared with 363 publicly available E. rhusiopathiae strains, as well as 13 strains of other Erysipelothrix species. Four strains tested represented serotypes 2 and 5 and the MLST groups ST 4, 32, 242, and 243. Their assembled circular genomes ranged from 1.8 to 1.9 kb with a GC content of 36–37%; a small plasmid was detected in strain 1023. Strains 1023 and 267 were multidrug-resistant. The resistance genes detected in the genome of strain 1023 were erm47, tetM, and lsaE-lnuB-ant(6)-Ia-spw cluster, while strain 267 contained the tetM and ermB genes. Mutations in the gyrA gene were detected in both strains. The tetM gene was embedded in a Tn916-like transposon, which in strain 1023, together with the other resistance genes, was located on a large integrative and conjugative-like element of 130 kb designated as ICEEr1023. A minor integrative element of 74 kb was identified in strain 1012 (ICEEr1012). This work contributes to knowledge about the characteristics of E. rhusiopathiae bacteria and, for the first time, reveals the occurrence of erm47 and ermB resistance genes in strains of this species. Phage infection appears to be responsible for the introduction of the ermB gene into the genome of strain 267, while ICEs most likely play a key role in the spread of the other resistance genes identified in E. rhusiopathiae. Full article

Background and Objectives: Enterococcus faecalis (E. faecalis) is a primary pathogen responsible for dental abscesses, which cause inflammation and pain when trapped between the crown and soft tissues of an erupted tooth. Therefore, this study aims to use specific phages as an alternative method instead of classical treatments based on antibiotics to destroy multidrug-resistant E. faecalis bacteria for treating dental issues. Materials and Methods: In the current study, twenty-five bacterial isolates were obtained from infected dental specimens; only five had the ability to grow on bile esculin agar, and among these five, only two were described to be extensive multidrug-resistant isolates. Results: Two bacterial isolates, Enterococcus faecalis A.R.A.01 [ON797462.1] and Enterococcus faecalis A.R.A.02, were identified biochemically and through 16S rDNA, which were used as hosts for isolating specific phages. Two isolated phages were characterized through TEM imaging, which indicated that E. faecalis_phage-01 had a long and flexible tail, belonging to the family Siphoviridae, while E. faecalis_phage-02 had a contractile tail, belonging to the family Myoviridae. Genetically, two phages were identified through the PCR amplification and sequencing of the RNA ligase of Enterococcus phage vB_EfaS_HEf13, through which our phages shared 97.2% similarity with Enterococcus phage vB-EfaS-HEf13 based on BLAST analysis. Furthermore, through in silico analysis and annotations of the two phages’ genomes, it was determined that a total of 69 open reading frames (ORFs) were found to be involved in various functions related to integration excision, replication recombination, repair, stability, and defense. In phage optimization, the two isolated phages exhibited a high specific host range with Enterococcus faecalis among six different bacterial hosts, where E. faecalis_phage-01 had a latent period of 30 min with 115.76 PFU/mL, while E. faecalis_phage-02 had a latent period of 25 min with 80.6 PFU/mL. They were also characterized with stability at wide ranges of pH (4–11) and temperature (10–60 °C), with a low cytotoxic effect on the oral epithelial cell line at different concentrations (1000–31.25 PFU/mL). Conclusions: The findings highlight the promise of phage therapy in dental medicine, offering a novel approach to combating antibiotic resistance and enhancing patient outcomes. Further research and clinical trials will be essential to fully understand the therapeutic potential and safety profile of these bacteriophages in human populations. Full article

Staphylococcus aureus is a versatile pathogen that does not only occur in humans but also in various wild and domestic animals, including several avian species. When characterizing S. aureus isolates from waterfowl, isolates were identified as atypical CC133 by DNA microarray analysis. They differed from previously sequenced CC133 strains in the presence of the collagen adhesin gene cna; some also showed a different capsule type and a deviant spa type. Thus, they were subjected to whole-genome sequencing. This revealed multiple insertions of large regions of DNA from other S. aureus lineages into a CC133-derived backbone genome. Three distinct strains were identified based on the size and extent of these inserts. One strain comprised two small inserts of foreign DNA up- and downstream of oriC; one of about 7000 nt or 0.25% originated from CC692 and the other, at ca. 38,000 nt or 1.3% slightly larger one was of CC522 provenance. The second strain carried a larger CC692 insert (nearly 257,000 nt or 10% of the strain’s genome), and its CC522-derived insert was also larger, at about 53,500 nt or 2% of the genome). The third strain carried an identical CC692-derived region (in which the same mutations were observed as in the second strain), but it had a considerably larger CC522-like insertion of about 167,000 nt or 5.9% of the genome. Both isolates of the first, and two out of four isolates of the second strain also harbored a hemolysin-beta-integrating prophage carrying “bird-specific” virulence factors, ornithine cyclodeaminase D0K6J8 and a putative protease D0K6J9. Furthermore, isolates had two different variants of SCC elements that lacked mecA/mecC genes. These findings highlight the role of horizontal gene transfer in the evolution of S. aureus facilitated by SCC elements, by phages, and by a yet undescribed mechanism for large-scale exchange of core genomic DNA. Full article