Bacteriophages, or phages, are viruses that infect and lyse bacteria, and can be found in soil, water, and even food. They are now recognized as playing fundamental regulatory roles in microbial ecology and as the most effective candidates for combating antibiotic resistance (AMR; Batinovic et al., 2019; Harada et al., 2018). Since Twort (1915) and d'Herelle (1917) discovered them, phages have been found in every environment inhabited by bacteria, with a ratio of at least 10 phage particles per bacterial cell, making them more diverse than bacteria (Clokie et al., 2011). The global abundance of bacteriophages is estimated to exceed 10³¹, making them the most prevalent life forms on Earth (Bisen et al., 2024; Williamson et al., 2005, 2003). In terrestrial environments, phage concentrations in soil reach approximately 10⁸ particles per gram, and similarly high abundances are reported in aquatic systems, approaching 10⁷ particles per milliliter (Filee et al., 2005; Breitbart et al., 2004). Due to their remarkable abundance and diversity, phages play an essential role in aquatic ecosystems at multiple levels. They contribute to regulating bacterial populations, shape microbial diversity, and influence bacterial evolution and pathogenicity (Canchaya et al., 2003; Miao and Miller, 1999). Additionally, phages contribute to gene flow through horizontal gene transfer, thereby affecting bacterial evolution and adaptation (Canchaya et al., 2003).

There is a resurgence of interest in bacteriophage research as highly specific antibacterial agents, driven largely by the global increase in multidrug-resistant (MDR) bacteria and the limitations of conventional antibiotics (World Health Organization, 2024). Phage intervention presents a highly specific, self-amplifying, and ecologically safe alternative for addressing bacterial infections resistant to chemical antimicrobials (Ali et al., 2023). This specificity also makes them of interest as biocontrol agents for environmental management, as they selectively kill specific bacteria, thereby restoring ecological balance without harming beneficial communities (Harada et al., 2018).

Members of the Pseudomonadaceae family, and specifically the genus Pseudomonas, are ubiquitous in nature and include both environmental degraders and opportunistic human pathogens. Members of the Pseudomonas alcaligenes group are aerobic Gram-negative bacilli distinguished by their metabolic versatility and ability to degrade pollutants such as aromatic hydrocarbons (Pan et al., 2021). Although generally regarded as non-pathogenic, recent clinical reports have associated P. alcaligenes and its close relative, P. paralcaligenes, with bloodstream and wound infections, and have occasionally reported carbapenem-resistant plasmids (Suzuki et al., 2013; Ono et al., 2023). Because members of the P. alcaligenes group are taxonomically and physiologically related to P. aeruginosa, they may serve as environmental reservoirs for resistance determinants and virulence-associated genes. Consequently, investigating bacteriophages that target members of the P. alcaligenes group contributes to understanding phage–host interactions within environmentally relevant Pseudomonas lineages.

Urban, manmade ponds represent engineered microecosystems characterized by variable temperatures, pH levels, salinity, and nutrient inputs (Subramanian, 2024). These systems can harbor diverse microbial communities, including bacteriophages, often subjected to environmental stress shaped by local environmental conditions (2021; Topka et al., 2019). Despite increasing interest in phage ecology, bacteriophage diversity in arid and semi-arid urban aquatic environments remains insufficiently characterized.

Recent developments in genomics have revolutionized the characterization of bacteriophages, thereby enhancing our understanding of their structural organization and evolutionary relationships (Shen and Millard, 2021). High-throughput sequencing has enabled the identification of new genes, including predicted open reading frames (ORFs) with undefined functions that may point to unexplored molecular tools. Notably, the identification of hypothetical proteins in phage genomes reflects the current underrepresentation of closely related phages in public databases and highlights gaps in functional annotations (Sanz-Gaitero et al., 2021). Similarly, analysis of tRNA content can provide insight into phage-host translational interactions. Phages that encode few or no tRNAs depend on the host's translational machinery, consistent with compact genome architectures reported for many lytic bacteriophages (Bailly-Bechet et al., 2007).

Prior research has characterized bacteriophages infecting Pseudomonas aeruginosa (Wang et al., 2023) and members of the P. alcaligenes group (Ulrich et al., 2024), uncovering substantial genetic diversity. Despite this growing body of work, phage diversity in urban aquatic environments within arid regions remains relatively understudied. Investigating these ecosystems provides an opportunity to identify bacteriophages with distinct structural and genomic features.

In this study, we reported the isolation and genomic characterization of Pseudomonas phage Amjad_SA, a lytic bacteriophage recovered from a manmade pond in Riyadh, Saudi Arabia, and active against a Pseudomonas isolate assigned to the P. alcaligenes group. We combined biological characterization, including host-range assessment, adsorption kinetics, one-step growth analysis, and stability under varying pH and temperature conditions, with genome annotation and phylogenetic analyses. Collectively, these data provide a detailed biological and genomic profile of phage Amjad_SA and contribute to the growing body of knowledge on Pseudomonas-infecting tailed bacteriophages isolated from arid urban aquatic environments.

In a sterile 15 mL tube, we mixed 3.0 mL of the host strain P. alcaligenes overnight culture (OD600 0.5) with equal volumes of each pond sample filtrate and 6 mL of double-strength LB (2 × LB) broth supplemented with 10 mM calcium chloride. The mixture was incubated at 30 °C overnight with shaking at 140 rpm. After incubation, we centrifuged the phage-infected cultures at 5,000 rpm for 20 min to remove excess residual material. We then decanted the supernatant into a sterile tube and filtered it through a 0.45 μm syringe filter to eliminate any remaining bacterial cells. We then added chloroform to the filtrate (1:5), mixed by vortex, and centrifuged at 3,000 rpm for 20 min. After removing the upper aqueous layer, the resulting solution is called the lysate. To verify the presence of the lytic phage in the lysate, a spot assay was conducted on P. alcaligenes using the soft agar overlay technique24 to assess the phage's lytic activity against the host. First, 5 mL of semi-solid LB broth (0.7% agar) was mixed with 200 μL of the P. alcaligenes culture (OD600 0.5) in a sterile tube. This mixture was then poured onto solid LB agar plates (1.7%) and allowed to solidify. After solidification, 10 μL aliquots of the isolated phage lysate were spotted onto the surface of the semi-solid LB agar in Petri dishes, which were then incubated at 30 °C. Plaque presence was evaluated after overnight incubation. Plates with plaques were considered positive for the lytic phage.

The double agar layer method was used to titrate the phage. First, we mixed 0.1 mL of the phage lysate with 50 μL of the host overnight culture suspension in a sterile 15 mL tube. We then gently vortexed the mixture and incubated it at room temperature for 10 min to promote phage adsorption to the bacterial host. Following this incubation, 5 mL of pre-warmed soft LB agar (0.7% agar) was added to the mixture. The mixture was mixed thoroughly and poured onto an LB agar plate (1.7% agar). This preparation was allowed to set at room temperature for 30 min to solidify, then incubated at 30 °C overnight. After the incubation period, the plates were examined for the presence of plaques, characterized by clear zones on the agar surface. Plates were deemed positive if any plaques were observed. The number of plaques was counted and recorded as plaque-forming units (pfu/mL; Twest and Kropinski, 2009).

A high-titer phage lysate (5 × 10¹¹ PFU mL⁻¹) was added (4 μL) to a copper mesh grid (Carbon Type-B, 200, Tedpella) and subjected to negative staining using 2% (w/v) aqueous uranyl nitrate at pH 5.0. The samples were analyzed using a TEM (JEM-1400, JOEL, Tokyo, Japan) at 100 kV. Images were captured at magnification levels ranging from × 80,000 to × 100,000. Digital recordings were made with a 10.7-megapixel CCD camera (4008 × 2664 pixels, QUEMESA, EMSIS) and iTEM software (Kenny et al., 2004). The morphological characteristics defined by the International Committee on Taxonomy of Viruses (ICTV) were used to evaluate the phenotypic variation of the bacteriophages (Turner et al., 2021; Ackermann, 2011).

The multiplicity of infection (MOI) was calculated based on the absolute numbers of bacteriophage particles and host bacterial cells present at the time of infection. The host culture (Pseudomonas alcaligenes group isolate 4L) was adjusted to 2.7 × 10⁹ CFU mL⁻¹, as determined by viable plate counts at the time of the experiment. The phage lysate (Amjad_SA) used for infection had a titer of 1.17 × 10¹¹ PFU mL⁻¹, determined prior to mixing. For infection assays, 100 μL of bacterial suspension was mixed with 100 μL of phage suspension. Because equal volumes were used, the MOI was calculated directly from the ratio of PFU mL⁻¹ to CFU mL⁻¹, corresponding to an MOI of approximately 40. This MOI was selected to ensure efficient and synchronous infection for adsorption and one-step growth experiments (Kutter and Sulakvelidze, 2004).

To determine the adsorption rate of the phage Amjad_SA to the host bacterium, the phage was mixed with P. alcaligenes group isolate 4L at the calculated MOI and incubated at 30 °C. For 20 20-min periods, 100 μL of the mixture was collected at 2-min intervals, then diluted with 0.9 mL of LB. The mixture was centrifuged at 5,000 rpm for 15 min. The supernatant containing the unabsorbed free phages was diluted 9-fold. The phage titers were determined using the double-layer agar plate plaque assay. The adsorption rate was measured as the percentage of free, unabsorbed phages in the culture system (Shao and Wang, 2008). The experiment was performed using three independent biological replicates. Mean values and standard deviations were calculated and are reported.

A one-step growth experiment was performed to determine the replication dynamics of phage Amjad_SA. Following adsorption, 7 mL of the host bacterium P. alcaligenes group isolate 4L culture was infected with phage Amjad_SA at the optimal MOI. The mixture was then incubated at 30 °C for 5 min. The mixture was then centrifuged at 5,000 rpm for 5 min to remove free, unabsorbed phages. The pellet was resuspended in 7 mL of LB broth and incubated at 30 °C. During the 100-min period, samples were taken at 10-min intervals. Each collected aliquot was diluted and counted using a double-layer agar plate plaque assay (Hyman and Abedon, 2009).

Phage stability under different pH and temperature conditions was assessed using a plaque assay. For thermal stability, the phage Amjad_SA lysate was divided into six aliquots and incubated at 15, 25, 35, 45, 55, and 65 °C at pH 7 for 6 h. For pH stability, the phage Amjad_SA lysate aliquots were incubated in SM buffer at pH 3, 5, 7, 9, and 11 at 30 °C for 6 h. Following the incubation period, the phages in each aliquot were subsequently titrated by the double agar layer plaque assay (Adams, 1959). However, these assays were performed as single-endpoint measurements and were not designed to assess long-term decay kinetics.

The lytic spectrum was tested against all bacterial strains in Table 1 using spot assays against a panel of Gram-negative and Gram-positive bacterial strains. We prepared double-layer agar plates, each containing a lawn of a different bacterial strain. We spotted 10 μL of the phage Amjad_SA lysate onto each soft-agar lawn of each strain. All assays were incubated overnight at 30 °C, corresponding to the temperature used for phage isolation, and checked for the presence of clear zones (Ackermann, 2011). The temperature was selected to maintain consistent experimental conditions and to avoid potential temperature-dependent effects on phage adsorption and replication. All tested bacterial strains are capable of growth at 30 °C under laboratory conditions. The presence of plaques (clear zones) indicated susceptibility, while the absence of plaques indicated resistance.

The environmentally isolated Pseudomonas alcaligenes group strain (isolate 4L) used for phage isolation served as the primary host for host-range testing. Additional P. alcaligenes strains were not included due to the limited availability of well-characterized reference isolates accessible during the study. Accordingly, the host-range analysis was designed to assess species-level specificity across the tested bacterial panel rather than intraspecies variation within P. alcaligenes.

In this study, we report the isolation and characterization of Pseudomonas phage Amjad_SA, a lytic bacteriophage recovered from a manmade pond in Riyadh, Saudi Arabia, and active against a Pseudomonas isolate assigned to the P. alcaligenes group. By integrating biological characterization with genomic and phylogenetic analyses, this work provides a comprehensive profile of phage Amjad_SA and contributes to the expanding dataset of Pseudomonas-infecting tailed bacteriophages recovered from urban aquatic environments within arid regions.

Urban artificial ponds in arid regions are exposed to variable physicochemical conditions, including fluctuations in temperature and pH, which can influence microbial community structure. Under the experimental conditions tested, phage Amjad_SA retained infectivity following exposure to temperatures between 25 and 65 °C and across a pH range of 5–9. These results indicate tolerance to the tested temperature and pH ranges; however, longer-term decay kinetics, repeated exposure experiments, and assessment of additional environmental parameters would be required to evaluate environmental persistence or to infer underlying stability mechanisms.

Phage Amjad_SA exhibited a narrow host range, infecting only the Pseudomonas isolate used for propagation among the strains examined. Such specificity is consistent with patterns commonly reported for tailed bacteriophages and is generally attributed to host receptor compatibility rather than broad host adaptability. Although host-range restriction has been linked to structural determinants such as tail fibers or baseplate components in other phages, no direct structural or functional evidence was obtained in this study to support specific receptor-binding mechanisms.

Kinetic analyses revealed rapid adsorption, with most virions attaching to host cells within 14 min, and a latent period of approximately 20 min, indicating efficient infection dynamics under the experimental conditions. The estimated burst size (590 ± 140 PFU per infected cell) was comparatively high relative to values reported for several Pseudomonas-infecting phages, suggesting productive viral replication. Variation in burst size is known to be influenced by host physiology and experimental parameters, and therefore these kinetic characteristics likely reflect both intrinsic phage properties and host–environment interactions. Collectively, these findings provide insight into the replication efficiency of phage Amjad_SA and underscore its capacity for effective propagation in the tested system.

Genomic analysis showed that Amjad_SA possesses a linear double-stranded DNA genome of 45.8 kb encoding 72 predicted open reading frames. Conserved genes involved in DNA replication, genome packaging, and virion assembly were identified, including terminase subunits, structural proteins, and an endolysin, indicating a typical modular organization of tailed bacteriophages. A substantial proportion of ORFs lacked confident functional annotation, a feature frequently observed in newly characterized bacteriophages and reflective of the limited representation of closely related phage genomes in current databases. These findings contribute additional genomic data to the growing collection of Pseudomonas-infecting bacteriophages recovered from urban aquatic environments.

The detection of a single tRNA gene (tRNA-Ser [aga]) suggests a reliance on the host translational machinery, a characteristic commonly reported for lytic phages with compact genomes. Such streamlined genomic architectures are consistent with evolutionary strategies that favor replication efficiency and reduced metabolic burden. Similar patterns have been documented across diverse bacteriophage lineages, in which limited tRNA repertoires support efficient infection cycles (Salmond and Fineran, 2015). Although the functional significance of the identified tRNA gene cannot be fully resolved based on genomic data alone, its presence may reflect adaptive optimization of translational processes during phage replication.

Comparative genomic and phylogenetic analyses placed Amjad_SA within a clade of Pseudomonas-infecting tailed bacteriophages, while highlighting genomic distinctions at the level of gene organization and sequence similarity. These differences underscore the genetic diversity present among phages infecting closely related bacterial hosts. However, comparative interpretations were limited to available reference genomes and should be refined as additional Pseudomonas phage sequences become available.

Comparative genomic analysis showed that phage Amjad_SA shares sequence similarity with Sinorhizobium phage phiLM21 while exhibiting distinct gene organization and variation within structural modules. Phylogenetic analyses based on whole-genome comparisons and terminase large subunit sequences placed Amjad_SA within a distinct clade of tailed bacteriophages infecting Pseudomonas. These findings highlight genomic diversity among phages infecting closely related bacterial hosts and underscore the contribution of Amjad_SA to the expanding catalog of Pseudomonas-infecting bacteriophages recovered from arid urban aquatic environments.

This study provides a framework for future investigations into the biology and genomics of phage Amjad_SA. Additional work incorporating expanded host panels, receptor-binding studies, proteomic analyses, and functional characterization of hypothetical proteins will be required to better resolve the molecular determinants of host specificity and infection dynamics. Such efforts will contribute to a broader understanding of phage diversity and functional variation among Pseudomonas-associated bacteriophages.

Several limitations of this study should be acknowledged. Host identification was based on 16S rRNA gene analysis and did not include whole-genome sequencing or multilocus sequence typing. Environmental parameters beyond temperature and pH were not measured, limiting ecological interpretation. In addition, functional predictions relied primarily on sequence similarity and automated annotation pipelines, and experimental validation of predicted gene functions was not performed. Future studies incorporating expanded host collections, refined genomic annotation approaches, and targeted functional assays will be required to further elucidate the biology of phage Amjad_SA.

This study presents the isolation and detailed biological and genomic characterization of Pseudomonas phage Amjad_SA, a lytic bacteriophage recovered from an urban artificial pond in Riyadh, Saudi Arabia, and active against a Pseudomonas isolate assigned to the P. alcaligenes group. Morphological analysis by transmission electron microscopy revealed an icosahedral head and a contractile tail, consistent with tailed bacteriophages of the class Caudoviricetes. Biological assays demonstrated efficient infection dynamics and a narrow host range under the experimental conditions tested.

Genomic analysis showed that Amjad_SA possesses a linear double-stranded DNA genome of 45,788 bp with a GC content of 60.4%, encoding 72 predicted open reading frames and a single tRNA-Ser (aga) gene. Conserved genes involved in replication, genome packaging, virion assembly, and host lysis were identified, alongside a substantial proportion of hypothetical proteins, reflecting current gaps in functional annotation for Pseudomonas-infecting phages. Importantly, no genes associated with known antibiotic resistance determinants or bacterial virulence factors were detected based on sequence similarity searches. Comparative and phylogenetic analyses placed Amjad_SA among related tailed bacteriophages and indicated closest similarity to Sinorhizobium phage phiLM21, while also revealing differences in gene organization and sequence similarity that distinguish Amjad_SA from available reference genomes.

Overall, this work expands the available biological and genomic data on Pseudomonas-infecting tailed bacteriophages isolated from urban aquatic environments within arid regions. The findings provide a foundation for future studies aimed at refining functional annotation, elucidating phage–host interactions, and further contextualizing bacteriophage diversity associated with Pseudomonas species. Collectively, this study delivers a well-resolved biological and genomic dataset for phage Amjad_SA and contributes to the reference framework for Pseudomonas-infecting tailed bacteriophages from understudied urban aquatic environments within arid regions.