Background Multidrug-resistant (MDR) pathogens such as Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae pose significant challenges to infection control and environmental sanitation, particularly in regions with high antimicrobial resistance (AMR) burdens. In 2021, Kenya reported approximately 28,500 deaths attributed to AMR, with K. pneumoniae accounting for the highest toll at 5220 deaths, followed by P. aeruginosa with 2,690, according to the Institute of Health Metrics. These pathogens are frequently detected in wastewater and hospital effluents, contributing to environmental persistence and transmission. Objective To isolate and descriptively characterise lytic bacteriophages (phages) targeting high-priority MDR bacteria from wastewater sources in Kilifi County, Kenya, with emphasis on genomic features that inform their safety, taxonomy, and potential relevance for future evaluation in applied contexts. Methods Four lytic phages were isolated using enrichment techniques against environmental MDR strains and purified by plaque assays. Whole-genome sequencing was performed using Oxford Nanopore technology, followed by annotation and comparative genomic analysis to determine taxonomic placement, conserved domains, and safety profiles. Results All four phages belong to the class Caudoviricetes and have genomic features consistent with obligately lytic replication, including the absence of lysogeny-associated genes and conserved structural modules. Comparative genomic analysis revealed global relatedness to previously described lytic phages. Preliminary spot-test observations demonstrated lytic activity against a limited set of local MDR isolates, providing initial descriptive evidence of their biological activity. Conclusion This study provides a genomics-based, preliminary characterisation of four wastewater-derived lytic phages. While their genomic profiles and observed activity suggest potential relevance for future evaluation in environmentally associated systems, further functional validation and application-orientated studies are required.
Emerging phytopathogens are a growing threat to global agriculture, undermining crop yields and jeopardizing food security. Among these, Pseudomonas coronafaciens pv. garcae (Pcg) causes coffee halo blight, a disease with serious implications for coffee production. Current control measures rely heavily on copper-based compounds and antibiotics like kasugamycin, both of which present environmental hazards and drive resistance development. In this context, bacteriophages offer a targeted and sustainable alternative. Although phage therapy has been explored for several plant pathogens, no phage-based solution has yet been tailored to control Pcg. Here, we describe an in-depth genomic characterization of four novel lytic phages (PCG-05T, PCG-06T, PCG-07T, and PCG-09T) capable of infecting Pseudomonas coronafaciens pv. garcae. Genomic sequencing and structural analysis via transmission electron microscopy revealed that phages PCG-05T, PCG-07T, and PCG-09T exhibit myovirus morphology, while phage PCG-06T displayed a siphovirus morphotype. Beyond taxonomic classification, we investigated the intrinsic cyclizability of their genomes, integrating insights from DNA biophysics to explore how sequence-dependent mechanical properties influence phage virion assembly. Our findings suggest that DNA bendability, shaped by base pair composition and helical periodicity, may correlate with phage morphogenesis efficiency. This study not only broadens the repertoire of candidate phages for biocontrol of coffee plant pathogens but also introduces an innovative hypothesis linking genome mechanics to phage virion fitness. These insights lay the groundwork for environmentally friendly phage-based strategies to combat bacterial blight in coffee plant cultivation.
Stenotrophomonas maltophilia is an emerging opportunistic pathogen with high intrinsic antimicrobial resistance, motivating renewed interest in bacteriophages as ecological components of microbial communities, with potential applications in phage therapy. Here, we isolated four lytic phages infecting clinical S. maltophilia strains, recovered from hospital wastewater and generated complete genomes using long-read sequencing. Using comparative genomics, and phylogenomic analyses, we examined the genome architecture, functional gene content, and evolutionary relationships within the 146 currently available Stenotrophomonas phage genomes. The double-stranded DNA phages ranged in size from 56.3 to 74.8 kb and encoded 79–94 proteins, with no detectable virulence or antimicrobial resistance genes. Comparative analyses showed that all isolated phages represent novel species and genera. Three phages clustered within a divergent clade of the family Schitoviridae, whereas one phage was more closely related to the Casjensviridae family members. Host range assays revealed variable infectivity patterns across clinical isolates, indicating host-dependent infection dynamics. Additionally, the phages encoded diverse anti-defense systems that were unevenly distributed among lineages and reflected their evolutionary history. These results expand the genomic and taxonomic landscape of S. maltophilia bacteriophages and highlight lineage-specific evolutionary strategies shaped by host immunity and environmental pressure.
Bacterial blight of coffee caused by Pseudomonas coronafaciens pv. garcae (Pcg) poses a significant threat to coffee production. This study reports the isolation and characterization of four lytic bacteriophages - PCG-05T, PCG-06T, PCG-07T, and PCG-09T - as potential biocontrol agents against Pcg. Isolated using multiple Pcg strains, the phages exhibited differential host specificity, plaque morphology, and lytic efficiency. Adsorption kinetics and one-step growth analyses revealed fast binding (PCG-06T and PCG-07T) and high burst sizes (PCG-05T and PCG-09T). In addition, phages PCG-06T and PCG-09T also displayed broad host range and strong lytic activity, with low frequencies of emergence of bacterial resistant mutants (≤10−3), with all phages positioning themselves as promising candidates for phage therapy. Electrostatic and diffusion analyses suggested that adsorption efficiency was influenced by surface charge interactions, with PCG-06T showing enhanced binding due to a higher (less negative) Zeta potential. In vitro inactivation assays demonstrated MOI-dependent suppression of Pcg populations, with phage cocktails yielding superior reductions - up to 6.17 log CFU/mL - especially at MOI 10000. Ex planta assays on artificially contaminated coffee plant leaves further confirmed significant bacterial inactivation, achieving up to 4.08 log CFU/mL reduction after 36 h. Despite slight regrowth at later time points, phage persistence remained stable, highlighting the potential of high-MOI applications for effective short-term control. Genomic screening confirmed the absence of undesirable genes, reinforcing safety for agricultural use. While results are promising, further studies on formulation, environmental stability, and field efficacy are needed to optimize phage-based interventions for controlling Pcg in coffee plantations.
Bacteriophages are increasingly recognized as key regulators of microbial ecosystems, yet their ecological role in fermented beverages remains insufficiently characterized. This review explores phage dynamics during cider fermentation, positioning cider as an emerging model for studying interactions between virulent and temperate phages in situ. Classical ecological models, including Kill-the-Winner (KtW) and Piggyback-the-Winner (PtW), describe contrasting viral strategies, where lysogeny is favored either under low host density or within dense microbial communities. However, studies from marine, terrestrial, and gut ecosystems indicate that these infection modes coexist along a dynamic continuum rather than as discrete outcomes.Cider fermentation, marked by acidification (pH of 3.5 - 4.3), ethanol accumulation (1.2 - 8.0 %, v/v), and high microbial density, likely supports both lytic and lysogenic infections depending on the fermentation stage and technological practices. Despite this potential, no longitudinal studies have yet examined the temporal dynamics of phage life cycles in cider. Furthermore, climate change may indirectly modulate phage–bacteria interactions by altering fruit physiology, epiphytic microbiota, and fermentation kinetics – an aspect rarely considered in current phage ecology. Finally, prophages may provide adaptive benefits through auxiliary metabolic genes (AMGs), which could enhance bacterial stress tolerance and nutrient utilization. These mechanisms highlight temperate phages as potential contributors to microbial adaptation and ecosystem stability in fermented environments.
The increasing complexity of virology has not been matched by equivalent access to experimental training, largely due to biosafety constraints, infrastructure demands, and the relatively high cost of practical virology compared to other areas of microbiology. These limitations are particularly pronounced in resource-constrained settings, where access to laboratory infrastructure is restricted. This imbalance has reduced hands-on learning opportunities and risks shifting virology education toward predominantly theoretical approaches. Bacteriophages provide a safe, cost-effective, and experimentally robust alternative for teaching fundamental virological principles. Their use enables direct observation of viral infection dynamics, quantitative analysis through plaque assays, and exploration of key concepts such as host specificity, viral replication, and lysogeny. Moreover, phage-based systems offer accessible entry points into contemporary topics, including antimicrobial resistance, phage therapy, viral evolution, and biotechnology applications. By integrating bacteriophages into virology curricula, educators can bridge the gap between theoretical knowledge and experimental practice while supporting scalable and inclusive models of training across diverse institutional contexts. Evidence from implementation in health-related programs in a middle-income country further highlights the feasibility and educational value of this approach in resource-constrained environments. Existing phage-based educational initiatives, particularly discovery-oriented programs such as SEA-PHAGES, further support the scalability of this approach and highlight its potential contribution to broader scientific literacy about viral emergence and epidemics.
Food safety challenges, the rapid emergence of antimicrobial resistance (AMR), and increasing zoonotic threats have intensified the need for innovative and sustainable antimicrobial strategies. Among these, bacteriophages have re-emerged as highly specific and self-amplifying agents for controlling bacterial pathogens. However, viral parasitism in microbial systems extends beyond bacteriophages alone. In this review, we present a comprehensive perspective on viruses infecting microbial hosts, including bacteriophages, cyanophages, mycoviruses (fungi-infecting viruses), protozoan viruses, and virophages. We emphasize their classification as obligate intracellular parasites, reflecting their dependence on host cellular machinery for replication and their complex infection dynamics. Key aspects of phage biology, including host recognition, lytic activity, and microbial regulation, are discussed alongside their ecological and functional roles. Beyond therapeutic applications, microbial viruses play significant roles in food safety, agriculture, aquaculture, and environmental management. These include applications in biocontrol, bacterial typing, biofilm disruption, and the management of multidrug-resistant pathogens such as Mycobacterium and Mycoplasma. Importantly, only specific bacteriophage-based products have been granted regulatory approval, highlighting the need for careful evaluation of safety and efficacy. Furthermore, specialized viral groups such as cyanophages and virophages contribute to microbial population dynamics, influence ecosystem stability, and provide valuable model systems for studying host–virus interactions and evolutionary processes. Collectively, this review highlights viruses infecting microbial hosts as versatile biological control, therapeutic, and environmental agents, offering promising avenues for addressing global challenges in health, agriculture, and sustainability.
The marine ecosystem harbors numerous opportunistic bacteria, among which Shewanella spp. has gained increasing attention due to its medical significance. The extensive use of antibiotics has led to the emergence of antibiotic-resistant bacteria; therefore, bacteriophages are being considered effective alternative therapeutic agents, although they remain poorly characterized. In this study, we isolated and broadly characterized a lytic bacteriophage, designated vB_SheP_NIOT1, against Shewanella spp. from seawater. The phage exhibited a non-enveloped icosahedral capsid with a long, flexible, non-contractile tail, with an approximate length of 210 nm. It displayed strong environmental tolerance and stable infectivity. The latent period was approximately 60 min, and the burst size was approximately 120 PFU per infected cell. Genomic characterization revealed that vB_SheP_NIOT1 has a 62 kb circular dsDNA genome with 49% GC content and encodes 107 predicted coding sequences, with no antimicrobial resistance genes, virulence factors, tRNAs, or mobile genetic elements. Comparative genomic analysis revealed a highly divergent nucleotide sequence and conserved proteomic genes. Taxonomic analysis using VIRIDIC, VICTOR, and vConTACT3 classified the phage into a candidate novel genus within Queuovirinae, rather than assigning it to any currently established genus.
Bacteriophages have been shown as promising adjuvants or alternatives to antibiotics in treatment of infections with Pseudomonas aeruginosa, considered by WHO as a “priority pathogen” due its capacity to develop antimicrobial resistance and to form antibiotic tolerant biofilms.The observed differences in biofilm-phage interactions need to be resolved to develop efficient and predictable phage-based antibacterial strategies. We have studied the phage- P. aeruginosa biofilm interactions focusing on the role of profage Pf4, quorum-sensing proficiency (QS) and biofilm metabolism. We have investigated two different lytic phages (NP3 and vB_Pae-TbilisiM32 (M32)) on P. aeruginosa biofilms of following strains: reference strains PA14, PAO1, MPAO1 lineage and a clinical cystic fibrosis isolate. The role of Pf4 prophage was explored by comparisons of the lytic phages efficacy on biofilms of PAO1 (MPAO1 lineage) and its ΔPf4 mutant. We have further inquired the role of AHL-dependent QS and Pseudomonas quinolone signal (PQS) in protection against lytic phages by investigating biofilms of lasR, rhlR, lasRrhlR as well as pqs (pqsC, pqsR, pqsH) PAO1 mutants. The role of the metabolic state for the effect of phage treatment in biofilms of different ages and after repeated phage treatment has been investigated. We document differences in P. aeruginosa biofilms in response to phage exposure and demonstrate the protective effect of prophage Pf4 and QS against lytic phages as well as the increased phage protection offered by low metabolic activity in older biofilms.
The discovery of bacteriophage one century ago by the French-Canadian Félix d'Herelle set off controversies as to the nature of bacteriophage as well as over the priority and credit for this discovery. The background and life of d'Herelle reveals a complex, self-taught outsider in science who was strongly influenced by his admiration of Louis Pasteur, but also his attachment to the philosophical positions of early 17th century philosophers, especially Francis Bacon. D'Herelle left substantial unpublished writings on his philosophical musings toward the end of his life.
We argue that a paradigm shift is needed in the analysis of phage DNA packaging. We then test a prediction of the following paradigm shift-engendering hypothesis. The motor of phage DNA packaging has two cycles: (1) the well-known packaging ATPase-driven (type 1) cycle and (2) a proposed back-up, shell expansion/contraction-driven (type 2) cycle that reverses type 1 cycle stalls by expelling accidentally packaged non-DNA molecules. We test the prediction that increasing the cellular concentration of all macromolecules will cause packaging-active capsids to divert to states of hyper-expansion and contraction. We use a directed evolution-derived, 3-site phage T3 mutant, adapted to propagation in concentrated bacterial cytoplasm. We find this prediction correct while discovering novel T3 capsids previously obscure.
Phages infecting Lactococcus lactis pose a serious threat to the dairy fermentation sector. Consequently, they are among the most thoroughly characterized Gram positive-infecting phages. The majority of lactococcal phages belong to the tailed family of phages named the Siphoviridae. The coliphage lambda and the Bacillus subtilis phage SPP1 have been the predominant comparators for emerging siphophages both genomically and structurally and both phages recognize a membrane protein receptor. In contrast, the lactococcal P335 group phage TP901-1 attaches to cell wall surface polysaccharides. It is a typical "lambdoid" siphophage possessing a long non-contractile tail and a genomic architecture reminiscent of lambda and SPP1 despite low or undetectable sequence homology in many of its encoded products, especially those involved in host recognition. A functional analysis of the structural components of TP901-1 was undertaken based on the characterization of a series of mutants in the region encoding the capsid and tail morphogenetic elements. Through this analysis, it was possible to deduce that, despite the lack of sequence homology, the overall genomic architecture of Siphoviridae phages typified by functional synteny is conserved. Furthermore, a model of the TP901–1 assembly pathway was developed with potential implications for many tailed phages.
The concept of bacteriophage multiplicity of infection (MOI) - ratios of phages to bacteria - historically has been less easily applied than many phage workers would prefer or, perhaps, may be aware. Here, toward clarification of the concept, I discuss multiplicity of infection in terms of semantics, history, mathematics, pharmacology, and actual practice. For phage therapy and other biocontrol purposes it is desirable, especially, not to solely employ MOI to describe what phage quantities have been applied during dosing. Why? Bacterial densities can change between bacterial challenge and phage application, may not be easily determined immediately prior to phage dosing, and/or target bacterial populations may not be homogeneous with regard to phage access and thereby inconsistent in terms of what MOI individual bacteria experience. Toward experiment reproducibility and as practiced generally for antibacterial application, phage dosing instead should be described in terms of concentrations of formulations (phage titers) as well as volumes applied and, in many cases, absolute numbers of phages delivered. Such an approach typically will be far more desirable from a pharmacological perspective than solely indicating ratios of agents to bacteria. This essay was adapted, with permission, from an appendix of the 2011 monograph, Bacteriophages and Biofilms, Nova Science Publishers.
ϕEf11, a temperate Siphoviridae bacteriophage, was isolated by induction from a root canal isolate of Enterococcus faecalis. Sequence analysis suggested that the ϕEf11 genome included a contiguous 8 gene module whose function was related to head structure assembly and another module of 10 contiguous genes whose products were responsible for tail structure assembly. SDS-PAGE analysis of virions of a ϕEf11 derivative revealed 11 well-resolved protein bands. To unify the deduced functional gene assignments emanating from the DNA sequence data, with the structural protein analysis of the purified virus, 6 of the SDS-PAGE bands were subjected to mass spectrometry analysis. 5 of the 6 protein bands analyzed by mass spectrometry displayed identical amino acid sequences to those predicted to be specified by 4 of the ORFs identified in the ϕEf11 genome. These included: ORF8 (predicted scaffold protein), ORF10 (predicted major head protein), ORF15 (predicted major tail protein), and ORF23 (presumptive antireceptor).
Phage therapy is a promising treatment of multi-drug resistant (MDR) bacterial infections but is limited by the narrow host range of phage. To overcome this limitation, we developed a host range expansion (HRE) protocol that expands the host range of Pseudomonas aeruginosa-specific phage by cycles of co-incubation of phage with multiple P. aeruginosa strains. Application of the HRE protocol to a mixture of 4 phages, using 16 P. aeruginosa strains for development, resulted in undefined phage mixtures with greatly expanded host range. Individual phage clones derived from the undefined mixture had expanded host ranges but no individual clone could lyse all of the strains covered by the undefined mixture from which it was isolated. Reconstituting host range-characterized clones into cocktails produced defined cocktails with predictable and broad host ranges. The undefined mixture from the 30th cycle of the mixed-phage HRE (4ϕC30) showed a dose-dependent ability to prevent biofilm formation by, and to reduce a pre-existing biofilm of, 3 P. aeruginosa clinical isolates that produced high amounts of biofilm. A defined cocktail reconstituted from 3 host range-characterized clones had activity on high biofilm-formers susceptible to the phage. Phage therapy was superior to antibiotic therapy (levofloxacin) in a strain of P. aeruginosa that was resistant to levofloxacin. The HRE protocol establishes a rapid approach to create libraries of phage clones and phage cocktails with broad host range, defined composition and anti-biofilm activity.
Phages infecting Lactococcus lactis pose a serious threat to the dairy fermentation sector. Consequently, they are among the most thoroughly characterized Gram positive-infecting phages. The majority of lactococcal phages belong to the tailed family of phages named the Siphoviridae. The coliphage lambda and the Bacillus subtilis phage SPP1 have been the predominant comparators for emerging siphophages both genomically and structurally and both phages recognize a membrane protein receptor. In contrast, the lactococcal P335 group phage TP901-1 attaches to cell wall surface polysaccharides. It is a typical "lambdoid" siphophage possessing a long non-contractile tail and a genomic architecture reminiscent of lambda and SPP1 despite low or undetectable sequence homology in many of its encoded products, especially those involved in host recognition. A functional analysis of the structural components of TP901-1 was undertaken based on the characterization of a series of mutants in the region encoding the capsid and tail morphogenetic elements. Through this analysis, it was possible to deduce that, despite the lack of sequence homology, the overall genomic architecture of Siphoviridae phages typified by functional synteny is conserved. Furthermore, a model of the TP901-1 assembly pathway was developed with potential implications for many tailed phages.
We have analyzed fecal bacterial and viral communities of a patient with recurrent C. difficile infection (rCDI) who was cured by fecal microbiota transplantation (FMT). The "Zürich Patient" experienced immediate cure and has remained free of symptoms for now over 5 y. Donor-similar bacterial compositions after 4.5 y post-FMT demonstrated sustainable engraftment of donor microbiota predominated by Bacteroidetes and Firmicutes bacteria. Appearance of beneficial species Faecalibacterium prausnitzii and Akkermansia municiphila was detected while disease-related Proteobacteria decreased. Stabilization of the microbiota took longer than expected from the rapidly improving clinical symptoms, suggesting the need for longer-lasting patient observation. The virome was mainly composed of Caudovirales bacteriophages but surprisingly also contained sequences related to a Chlorella giant virus that normally infects green algae not known to inhabitate the human intestine. FMT is highly effective against rCDI and is presently broadening its application to other conditions including inflammatory bowel disease (IBD). Here, we discuss the prospects and challenges of FMT against rCDI and other indications including a focus on bacteriophages.
We have developed a phagebiotic composition using 8 virulent bacteriophages (2 strains of each species) which are able to lyse Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus. The unique character of the developed composition is ensured by particular properties of each bacteriophage comprising the preparation, including their range of lytic activity toward specific bacterial pathogens, morphology of their plaques, cycle of their development, restriction profile of their DNAs, specificity of their genomes (based on complete genome sequencing), and other properties. The preparation did not produce any signs of acute or chronic intoxication in the experimental animals. Therapeutic and prophylactic efficiency of the phagebiotic composition was demonstrated in the prevention and treatment of the experimental acute K. pneumoniae infection in mice. The investigations have shown that the preparation possesses a high therapeutic efficiency and is highly competitive with ciprofloxacin which is very effective against the infective strain K. pneumoniae. Our small-scale clinical trial was aimed to evaluate therapeutic effectiveness of the phagebiotic composition in an epidemiological emergency situation in an intensive care unit, caused by multi-resistant strains of Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa. Seventy nine per cent of the initial samples from 14 patients' endotracheal aspirate, blood and urine were contaminated. Twenty-four hours after the 3-day phage therapy (20 ml of cocktail at a titer for each phage 108 pfu/ml were introduced intragastrically through a tube once a day) contamination level dropped to 21%. Hence the obtained results enabled us to create a new phagebiotic composition that may be used as an alternative to antibiotics to treat these healthcare-associated infections.
Xylella fastidiosa subsp. fastidiosa (Xff) is the causal agent of Pierce's Disease (PD) of grapevines and is vectored by the glassy-winged sharpshooter (GWSS, Homalodisca vitripennis). Previously we have reported the development of a bacteriophage (phage) based biocontrol system for PD, but no information on insect transmission of phages has been reported. Here we communicate that laboratory reared GWSSs fed on cowpea plants (Vigna unguiculata subsp. unguiculata) harboring the virulent phage Paz were able to uptake of phage efficiently when the phage was present in high concentration, but were inefficient in transfer to plants.
