Cuong V. Hoang1Jonathan Fan1Lauren Bhasin1Anthony Del Mundo1Vanessa Law1Dominic Nguyen1Suraj Ganiger1Ashley Mansour1Christi L. McElheny2
Yohei Doi2,3,4
Olakunle I. Olawole1*- 1Department of Microbiology & Plant Pathology, University of California, Riverside, Riverside, CA, United States
- 2Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States
- 3Department of Microbiology, Fujita Health University School of Medicine, Toyoake, Aichi, Japan
- 4Department of Infectious Diseases, Fujita Health University School of Medicine, Toyoake, Aichi, Japan
Carbapenem-resistant Raoultella planticola (CRRP) is an emerging nosocomial pathogen with limited therapeutic options. Here, we describe the comparative characterization of two novel virulent bacteriophages, Macy and Sally, both isolated from the same soil microenvironment. Macy exhibits exceptional lytic potency, with a burst size of 8,375 PFU per infected cell, narrow host specificity, and pronounced biofilm-disrupting activity likely mediated by a putative depolymerase. In contrast, Sally displays a broader host range, infecting both R. planticola and R. ornithinolytica (including a clinical CRRP isolate), while maintaining moderate lytic activity, notable acid tolerance, and substantial biofilm reduction. SNP analysis revealed that resistant isolates carried mutations in genes linked to surface polysaccharide biosynthesis and LysR-family transcriptional regulation, conferring resistance at a measurable cost to bacterial growth fitness. Genomic and phylogenomic analyses further revealed distinct evolutionary trajectories: Macy is a large (147.8 kb) member of Straboviridae Straboviridae with a mosaic genome related to Raoultella phages, whereas Sally is a compact (48.5 kb) Casjensviridae phage that is siphovirus more closely aligned with Klebsiella and Enterobacter phages. Pangenomic comparisons highlighted Macy’s strain-specific gene expansions versus Sally’s cross-genus homology, emphasizing divergent adaptation strategies. Together, these findings illustrate the complementary therapeutic potential of Macy and Sally and establish a genomic and phenotypic foundation for developing effective phage cocktails against multidrug-resistant Raoultella infections.
Introduction
The rapid global rise of carbapenem-resistant Gram-negative bacteria represents a critical threat to public health, significantly restricting therapeutic options for the treatment of severe and life-threatening infections (Jean et al., 2022). These organisms are often associated with high morbidity and mortality rates, particularly in healthcare settings where immunocompromised and critically ill patients are at elevated risk. Among the array of emerging multidrug-resistant pathogens, Raoultella planticola has garnered increasing attention. Formerly classified within cluster II of the genus Klebsiella, R. planticola is a Gram-negative, facultatively anaerobic rod that has historically been considered an environmental or low-virulence organism. However, it is now increasingly implicated in opportunistic and hospital-acquired infections, including urinary tract infections, bacteremia, and pneumonia, particularly in patients with underlying comorbidities or invasive medical devices (Zhu et al., 2024; Zou et al., 2020; Wang et al., 2015; Xu et al., 2015). Of particular concern is the ability of R. planticola to acquire and harbor carbapenemase-encoding genes, including blaKPC, blaNDM, and blaOXA-48, which confer resistance to carbapenem antibiotics, often considered last-resort treatments for multidrug-resistant infections (Zou et al., 2022). The emergence and dissemination of carbapenem-resistant R. planticola (CRRP) strains not only complicate clinical management but also raise the risk of horizontal gene transfer to other pathogenic species, exacerbating the broader antimicrobial resistance crisis (Qu et al., 2015). This growing threat highlights an urgent need for enhanced surveillance, rapid diagnostic tools, and the development of novel antimicrobial agents and therapeutic strategies (Castanheira et al., 2009).
The primary means of managing R. planticola infections is by administering antibiotics; however, recent studies have shown high concerns about the rapid development of antibiotics resistant variants. While tigecycline-resistant R. planticola clinical isolates have been reported (Li et al., 2022), the majority of reported cases are carbapenem-resistant strains. Carbapenems are broad-spectrum antibiotics often us ed. as the last line of defense against serious bacterial infections, especially those caused by multidrug-resistant bacteria. However, CRRP strains have been reported in China (Qu et al., 2015; Xu et al., 2015; Liu et al., 2019; Chen et al., 2020; Zou et al., 2020; Li et al., 2022; Zou et al., 2022; Wang et al., 2023; Zhu et al., 2024), Taiwan (Tseng et al., 2014; Wang et al., 2015; Huang et al., 2018), Turkey (Atici et al., 2018; Ozbey et al., 2023), Germany (Schauer et al., 2022), and the United States (Castanheira et al., 2009; Atici et al., 2018; Park et al., 2019; Iovleva et al., 2020; Ballash et al., 2021; Ozbey et al., 2023). The rapid development of resistant variants has left R. planticola infections with very limited treatment options, with the consequence of prolonged hospital stays, increased risk of complications and spread of resistant bacteria in healthcare settings, posing a significant risk to other patients (Österblad et al., 2012; Pfeifer et al., 2012; Piedra-Carrasco et al., 2017). Therefore, urgent efforts are needed to develop new management strategies to prevent their further dissemination and mitigate the associated public health risks.
In light of this, bacteriophages offer a highly promising and potentially transformative approach to combating the growing threat of multidrug resistance, particularly among encapsulated pathogenic bacteria such as Raoultella species. Their inherent characteristics make them compelling therapeutic candidates. For instance, phages exhibit high specificity, targeting only specific bacterial strains, thereby minimizing disruption to the beneficial microbiota. They are also non-toxic to human cells and possess the ability to self-replicate at the site of infection, enhancing their therapeutic effect (Born et al., 2017; Hasanien et al., 2024; Wernicki et al., 2017). Moreover, their natural abundance in the environment also makes them readily available. These attributes collectively position phages as a potentially powerful tool for controlling and eradicating antibiotic-resistant bacterial infections, offering a much-needed alternative to traditional antibiotic therapies. Although multiple studies have demonstrated the effectiveness of lytic phages in targeting various serotypes of multidrug-resistant Klebsiella pneumoniae (Hung et al., 2011; Dufour et al., 2015; Anand et al., 2020; Dhungana et al., 2021; Hesse et al., 2021; Ichikawa et al., 2023; Weber-Dabrowska et al., 2023; Gholizadeh et al., 2024; Zhao et al., 2025), research on phage-based studies against Raoultella species remains extremely limited, with only a small number of phages reported and even fewer characterized in depth. Existing studies, including two phages isolated from R. ornithinolytica in Iran, provided initial isolation data but did not include mechanistic or in vivo evaluations needed to establish therapeutic potential (Zamani et al., 2019). Therefore, studies on phage therapy treatment against CRRP remains largely unexplored, and there is a need for well-characterized phages with demonstrated biofilm-disrupting and lytic activity against clinical isolates.
A major driver of multidrug-resistance and bacterial persistence across diverse environments is the biofilm, a community of surface-attached bacterial cells (Andrés-Lasheras et al., 2024). Their complex three-dimensional structure poses a significant challenge in both clinical and food industry settings, as conventional antibiotics struggle to penetrate biofilms embedded within an extracellular polymeric substances (EPS) matrix, thus making the search for effective biofilm eradication strategies crucial (Donlan, 2000). Recent research has highlighted the potential of phage-encoded enzymes, particularly endolysins and depolymerases, as promising alternatives (Hughes et al., 1998; Islam et al., 2024; Oliveira et al., 2019). Endolysins can directly degrade bacterial cell walls, causing rapid lysis, while depolymerases break down the EPS that protect biofilm communities, thereby enhancing phage penetration and bacterial clearance.
Despite the growing clinical recognition of R. planticola as an emerging opportunistic pathogen, including strains exhibiting carbapenem resistance, there remains a critical lack of targeted therapeutic options, and phage-based interventions for this genus are still poorly explored. Existing studies have identified Raoultella phages, yet their therapeutic potential has not been systematically evaluated, leaving a substantial gap in alternative treatments for multidrug-resistant infections. Therefore, the aim of this study was to isolate and characterize new lytic bacteriophages active against carbapenem-resistant R. planticola, assess their host range and biofilm-disrupting capacity, and evaluate their genomic suitability for therapeutic development. By establishing foundational biological and genomic profiles of these candidate phages, this work provides essential groundwork for the rational design of future therapeutic phage cocktails targeting CRRP.
Materials and methods
Bacterial and phage isolation
Soil samples were collected from the University of California, Riverside nematology greenhouse for bacterial and phage isolation. For bacterial isolation, soil samples were agitated in double-strength LB broth at 30 °C and 280 rpm for 2 h. Suspensions were serially diluted and plated on LB agar to obtain individual colonies. Bacterial isolates were initially identified based on colony morphology and confirmed by 16S rRNA gene sequencing. We focused on a Raoultella strain, which was further validated through whole-genome sequencing. This strain was subsequently used as the host for phage isolation. The remaining soil suspension was centrifuged at 7,000 rpm, and the supernatant was filter-sterilized using 0.22 μm pore filters to collect soil filtrates. Filtrates were enriched overnight with a cocktail of all isolated bacterial strains in a 250 mL flask at 30 °C and 280 rpm. After centrifugation (7,000 rpm) and filtration, the enriched supernatants were spotted onto overlays of each bacterial isolate and incubated overnight at 30 °C. Plaques showing clear lysis were picked and resuspended in phage SM buffer (50 mM Tris–HCl, 100 mM NaCl, 8 mM MgSO₄, 0.01% gelatin). Phages were further purified by three rounds of double-layer agar overlay on their respective hosts and stored at −80 °C for long-term preservation.
Antibiotics susceptibility
A two-fold serial dilution of antibiotic was prepared in sterile LB broth, spanning an appropriate concentration range. Each well of a 96-well plate contained 50 μL of antibiotic dilution and 50 μL of LB broth. A standardized bacterial suspension of R. planticola RP8, adjusted to a turbidity of 1.6, was added at 50 μL per well, resulting in a final well volume of 150 μL. Control wells included a growth control (bacteria without antibiotic) and a sterility control (LB only). Plates were incubated at 30 °C for 48 h with intermittent shaking in a SpectraMax iD3 multi-mode microplate reader (Molecular Devices, San Jose, CA, United States). Absorbance readings were collected every 30 min, and wells were visually inspected at the end of incubation to assess bacterial growth.
Host range assay
Multiple strains of R. planticola and R. ornithinolytica of clinical origin (Table 1) were used to evaluate phage host range. Bacterial lawns were prepared by mixing 100 μL of overnight culture, standardized to an OD₆₀₀ of 0.8, with 5 mL of molten 0.5% top agar, which was then overlaid on LB agar plates. High-titer phage suspensions (15 μL) were spotted onto the bacterial lawns. Plates were incubated at 28 °C, and lysis zones were recorded after overnight incubation.
Table 1. Lists of bacterial strains and their sensitivity to Macy and Sally.
Stability assays
For thermal stability assay, the Sally and Macy phage samples (106–107 PFU/mL) were incubated at 30, 40, 50, 60, 70 or 80 °C for 1 h in a thermocycler. Samples were collected at 6, 12, 18, and 24 h post-incubation for the time-course temperature stability test. For pH stability assay, SM buffer was standardized to different pH values (1, 3, 5, 7, 9, 11, or 13) using 1 M HCl or 1 M NaOH. A 10-fold serial dilution of each phage sample was made with the pH-standardized solutions and incubated at 37 °C for 1 h. Heat- and pH-treated samples were serially diluted and relevant dilutions were titered on double-layer agar overlays. Treatments were performed in triplicates and each experiment repeated at least twice.
Multiplicity of infection
Bacterial and phage titers (CFU/mL and PFU/mL, respectively) were used to prepare phage-bacterial suspensions at MOIs of 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001. Each MOI group was 10-fold serially diluted in LB broth and incubated at 30 °C for 5 h with shaking at 180 rpm. Lysates were subsequently serially diluted and titrated on double-layer agar overlays. All treatments were performed in triplicate, and experiments were repeated at least twice. Values are presented as the average of all collected readings. The MOI yielding the highest final phage titer was designated as optimal and used for all downstream applications.
One-step growth curve
To determine phage burst size, latent period, and burst time, phage-bacterial suspensions were prepared at the optimal MOI in 10 mL volumes. Suspensions were incubated at 30 °C for 10 min without shaking and then centrifuged at 10,000 rpm for 10 min. Pellets were resuspended in prewarmed 10 mL LB and incubated at 30 °C with shaking at 160 rpm. Samples were collected every 5 min for the first 50 min and every 10 min for an additional 100 min. Collected samples were serially diluted and titrated using double-layer agar overlays. All treatments were performed in triplicate, and experiments were repeated at least twice.
Phage adsorption rate assay
Phage-bacterial suspensions were prepared at the optimal MOI and incubated at 30 °C with shaking at 120 rpm. Samples (500 μL) were collected every 2 min for 20 min and centrifuged at 10,000 rpm for 2 min. The supernatant containing unadsorbed phages was filter-sterilized using 0.22 μm filters. Filtrates were serially diluted to determine the titer of free phages. All treatments were performed in triplicate, and experiments were repeated at least twice. The percentage of phage adsorption at different time points was calculated as: % Adsorption = (Initial PFU – free phage PFU) × 100/Initial PFU.
Phage lytic activity against planktonic cells and pre-formed biofilms
Phage-bacterial suspensions were prepared at MOIs of 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001 in a microtiter plate. Samples were incubated at 30 °C for 12 h with intermittent shaking in a SpectraMax iD3 multi-mode microplate reader (Molecular Devices, San Jose, CA, United States). Bacterial growth and media controls were included. Absorbance readings were collected every 30 min. All treatments were performed in triplicate and experiments were repeated at least twice. For biofilm assay, standardized bacterial cells (100 μL) were added to microtiter plate wells and incubated at 30 °C without shaking for 24 h to allow biofilm formation. After 24 h, 100 μL of 10-fold serially diluted phage stock, starting from 1010 PFU/mL, was added to pre-formed biofilms and incubated at 30 °C without shaking. After phage treatment, planktonic cells were removed by washing three times with sterile distilled water (SDW). Plates were air-dried, and biofilms were stained with 150 μL of 0.1% (w/v) crystal violet for 20 min at room temperature. Excess stain was removed by three gentle washes with SDW, and biofilms were air-dried, solubilized with 95% ethanol, and absorbance measured at 595 nm. Treatments were performed in triplicate, and experiments were repeated at least twice.
Assembly and annotation of phage genome sequence
Purified lysates of phages were concentrated by centrifugation, at 7000 g for 17 h at 10 °C. Phage genomic DNA was isolated using a Phage DNA isolation kit (Norgen Biotek; Thorold, Canada), and concentrations were measured using a QuBit DNA Quantification Kit (Life Technologies, Carlsbad, CA). Purified DNA was sequenced on the Illumina MiSeq platform using Illumina v2 500 cycle reagent chemistry, generating paired-end 250 bp reads (Steemers and Gunderson, 2005). Illumina reads were adapter- and quality-trimmed using Trimmomatic v0.39 (Bolger et al., 2014), and the quality of the trimmed reads was assessed using FastQC v0.11.71. High quality reads were pre-processed using FastX Toolkit2 and assembled using SPAdes 3.15.5 at k-mer settings of 21,33,55 (Bankevich et al., 2012). Contigs with low coverage were filtered from the assembly to yield a single contig for each phage. The quality and completeness of each genome assembly was evaluated using QUAST v5.1.0 (Gurevich et al., 2013) and BUSCO v5.8.0 (Seppey et al., 2019). The genomic termini of closed genome of Macy and Sally were analyzed using PhageTerm (Garneau et al., 2017). Phage genomes were annotated using Pharokka v1.3.0 (Bouras et al., 2023) followed by manual curation. Coding sequences (CDS) were predicted with PHANOTATE v1.5.1 (McNair et al., 2019) and tRNAs were predicted with tRNAscan-SE v2.0.11 (Chan et al., 2021).
Comparative genomic, phylogenetic and pangenome analysis of phage genomes
A comparative genome map was constructed using BLAST Ring Image Generator (BRIG), which is a cross-platform application that enables the interactive generation of comparative genomic images via a graphical user interface (Alikhan et al., 2011). The average amino acid identity (AAI) calculator tool (Assis et al., 2017) was used to compare identity between orthologous genes from characterized phages and reference phages. Orthologous genes were extracted, clustered to 30% protein identity and visualized using Clinker and clustermap.js (Gilchrist and Chooi, 2021). Whole genome and amino acid alignments were obtained using Mafft vs7.505 and trees were generated using Fasttree 2.1.11 with 3000 bootstrap replicates; the resulting phylogenies were visualized and annotated using FigTree vs 1.4.4 (Rambaut, 2008). For pangenome analysis, all selected phage genomes were annotated using Pharokka v1.3.0 (Bouras et al., 2023) followed by manual curation. Annotated files from Pharokka were used for a pan-genome analysis using Roary, which compiles orthogroups based on the presence or absence of orthogroup members (Page et al., 2015). The web-based tool, Venny vs 2.13 and UpSet (Lex et al., 2014) were used to quantitatively analyze intersecting gene sets between and within phage genera.
Transmission electron microscopy and halo phage plaque morphology
Electron microscopy of ultra-centrifuge-purified phage (~1 × 1010 PFU/ml) was performed by using phage dilutions made with a 5-fold dilution SM buffer. Diluted phage solutions were pipetted onto thin 400-mesh carbon-coated Formvar grids, stained with 2% (wt/vol) uranyl acetate, and air dried; excess stain solution was gently washed off with sterile distilled water. Specimen-containing grids were observed on a Talos L120C Transmission Electron Microscope operating at an acceleration voltage of 100 kV. To calculate mean values and standard deviations for dimensions of capsid and tail, three virions were measured and averaged, using Image J (Schneider et al., 2012). In addition to the TEM pictures, high dilutions that yielded 1–10 halo plaques were titered. Halo sizes were monitored at 24, 48, 72 and 98 h post-incubation. Pictures of plates were taken, and halo sizes were estimated using ImageJ. Image sizes were compared using students’ t-test. Each treatment was performed in triplicates.
Phage resistance and anti-phage defense mechanisms
Spontaneous phage-resistant mutants of R. planticola strain RP8 were generated by repeated exposure to high phage titers of Macy and Sally until resistant colonies emerged. Resistant isolates were purified by streaking and stored at −80 °C for downstream analyses. To assess potential fitness costs, growth dynamics of resistant and wild-type strains were monitored by measuring optical density (OD₆₀₀) every 30 min for 16 h using a SpectraMax iD3 microplate reader (Molecular Devices, USA) at 28 °C. Phage infections were conducted at a multiplicity of infection (MOI) of 0 and 100, where an MOI of 0 corresponds to a no-phage control. Genomic DNA from resistant and wild-type isolates was extracted using the Qiagen DNeasy Blood & Tissue Kit following the manufacturer’s instructions. DNA libraries were prepared and sequenced on the Illumina MiSeq platform to obtain paired-end reads. Raw reads were trimmed and quality-filtered, followed by de novo assembly using SPAdes v3.15. SNPs were identified using Snippy v4.6.04, aligning resistant strain reads to the wild-type genome. Variants unique to resistant strains were analyzed, and mutations in genes encoding surface-exposed or membrane-associated proteins were considered candidate phage receptors.
Statistical analyses
Quantitative data obtained from phage and bacterial plate titrations, microtiter plate absorbance readings, and ImageJ-based measurements were analyzed using GraphPad Prism software (version 9.3.1; GraphPad Software, San Diego, CA, United States). Bacterial growth data, measured as optical density (OD), were used to calculate the area under the growth curve (AUGC). For comparisons between two groups, unpaired two-tailed Student’s t-tests were performed. When unequal variances were detected, Welch’s correction was applied. For comparisons involving more than two groups, one-way analysis of variance (ANOVA) was conducted, followed by Tukey’s post hoc multiple comparisons test. A p-value of <0.05 was considered statistically significant.
Results
Isolation, morphology and host range
Four distinct bacteriophages were isolated from a soil sample collected near the Nematology greenhouse at the University of California, Riverside. Restriction digest analyses using EcoRV, EcoRI, BamHI, SmaI, and XhoI confirmed that the phages represented unique isolates (Data not shown). Naming was done in accordance with Texas A&M Center for Phage Technology policy, where novel phages are given names for mnemonic purposes. Two of these, designated Sally and Macy, were selected for detailed characterization because they exhibited distinct plaque morphologies and infection profiles compared to the other isolated phages. To evaluate phage activity, all experiments were conducted using the environmental R. planticola strain RP8 as the host. When propagated on overlay lawns of an environmental R. planticola strain RP8, Sally produced clear, circular lytic plaques (Figure 1C), while Macy generated clear plaques surrounded by expanding translucent halos (Figure 1A). The latter suggested the production of extracellular polysaccharide-degrading enzymes, potentially indicative of depolymerase activity and enhanced lytic efficacy. Transmission electron microscopy (TEM) revealed that both phages possess icosahedral capsids and long tails characteristic of the class Caudoviricetes. Macy displayed a compact icosahedral head (55.7 nm) and a shorter, contractile tail (78.6 nm), features consistent with Straboviridae (Myo-like) phages (Figure 1B, Supplementary Table S1). In contrast, Sally exhibited a markedly larger icosahedral head (99.4 nm) and a long, non-contractile tail (265.8 nm), aligning with the morphology of Casjensviridae (Sipho-like) phages (Figure 1D, Supplementary Table S1). Despite their shared overall architecture, these measurements highlight the pronounced structural divergence between the two phages. Host range analyses demonstrated genus-level specificity for both phages. Neither Sally nor Macy exhibited lytic activity against Klebsiella pneumoniae ATCC 13883 or Klebsiella oxytoca ATCC 49473 (Table 1). However, their virulence profiles within the Raoultella species differed. Macy infected only R. planticola RP8, suggesting a species-specific host range. In contrast, Sally displayed broader activity, infecting R. planticola RP8, and the clinical strains R. planticola ATCC 700831, R. ornithinolytica ATCC 31898, and Klebsiella oxytoca/Raoultella spp. (Table 1). Together, these results demonstrate that Sally and Macy represent distinct phages with contrasting host ranges and morphologies.
Figure 1. Morphological characteristics of phage Macy and Sally. (A,C) Plaques of Macy (A) and Sally (C) on the host R. planticola strain RP8. (B,D) Transmission electron microscope images revealing that Macy (B) belongs to the Straboviridae Myoviridae family and Sally (D) belongs to CasjensviridaeSiphoviridae.
Antibiotic susceptibility of R. planticola strain RP8
Whole genome sequencing of RP8, despite its environmental origin, revealed the presence of multiple antibiotics and multidrug resistance (MDR) genes (Table 2). Notable resistance genes included six copies of genes encoding multidrug efflux pumps, including multidrug resistance proteins B and MdtO, multiple antibiotic resistance protein MarB, and the MarR transcriptional regulator. Additional resistance determinants included genes for a bleomycin resistance protein, copper resistance protein, and an AraC family multidrug resistance transcriptional activator (Table 2). Therefore, we evaluated the susceptibility of RP8 to selected carbapenems, often considered the last line of defense against resistant infections. Bacterial growth in treated samples exceeded one-tenth of the no-treatment controls at the highest concentrations tested: 75 μg/mL for imipenem, 150 μg/mL for meropenem, and 187.5 μg/mL for doripenem (Figures 2A–C and Supplementary Figure S1). Such robust growth at these elevated antibiotic concentrations highlights the alarming risk of treatment failure and underscores the persistence of MDR infections even under intensive therapeutic pressure.
Table 2. Antibiotics and multidrug resistance genes identified in the genome of Raoultella planticola strain RP8.
Figure 2. Sensitivity of Raoultella planticola strain RP8 to Carbapenem group of antibiotics. Bacterial growth after treatment with different concentrations of Imipenem (A), Doripenem (B), and Meropenem (C) for 48 hours at 30°C using a plate reader.
Optimal multiplicity of infection
Determining the optimal multiplicity of infection (MOI) is essential for maximizing the efficacy of a phage in bacterial control applications. In this study, we evaluated the optimal MOI of Macy and Sally by infecting host bacterial cultures at varying MOIs ranging from 0.0001 to 100. We also assessed bacterial growth inhibition and phage replication efficiency by measuring optical density (OD600) and phage titer over time. Our results show that the optimal MOI for phage Macy is 0.001 (Table 3), while the optimal MOI for phage Sally is 1 (Table 4), as these conditions produced the highest burst sizes and most effective bacterial clearance without triggering excessive premature lysis that could limit phage propagation.
Table 3. Determination of optimal multiplicity of infection (MOI) of phage Macy.
Table 4. Determination of optimal multiplicity of infection (MOI) of phage Sally.
Lytic activity against planktonic and biofilm cells
Macy and Sally showed MOI-dependent lytic activity, with higher MOIs (1–100) driving faster bacterial reduction but limiting phage amplification due to rapid host depletion (Figures 3A–D). For Macy, an MOI of 0.1 provided an optimal balance with 65% lytic activity and a phage titer of 1.1 × 1010 PFU/ml, while for Sally, an MOI of 1 yielded 79% lytic activity and a titer of 1.5 × 109 PFU/ml. Percentage lytic activity refers to the proportion of bacterial growth suppressed in a phage-treated culture compared to an untreated control. Together, these results highlight phage-specific differences in infection dynamics and underscore the importance of optimizing MOI conditions to maximize bacterial suppression and phage propagation. Overall, Macy and Sally demonstrated strong lytic capacity, efficiently lysing planktonic R. planticola RP8 cells. For Macy, an MOI of 0.1 provided an optimal balance with 65% lytic activity and a phage titer of 1.1 × 1010 PFU/ml, while for Sally, an MOI of 1 yielded 79% lytic activity and a titer of 1.5 × 109 PFU/ml. Together, These results highlight phage-specific differences in infection dynamics and emphasize the need to tailor MOI conditions for maximizing both bacterial suppression and sustainable phage propagation. Together, these results demonstrate that Macy and Sally efficiently lyse planktonic R. planticola RP8 cells. Furthermore, Macy demonstrated significant disruption activity against pre-formed biofilm of the R. planticola strain RP8. When applied at different concentrations (107–109 PFU/ml), the phage effectively reduced biofilm biomass (Figure 4A), as quantified by crystal violet staining. Compared to untreated controls, phage-treated biofilms showed a 56-71% reduction (Figure 4B). Similarly, Sally exhibited strong biofilm disruption activity against pre-formed RP8 biofilms (Figures 4A–C) and reduced biofilm production by 82–85% (Figure 4D). Together, these findings highlight the potential of phages Macy and Sally as effective biocontrol agents for managing biofilm-associated infections and reducing surface contamination in both clinical and environmental settings.
Figure 3. Lytic activity of phage Macy and Sally against planktonic cells. (A,C) Bacterial growth curve (A) and corresponding area under growth (C) of phage Macy. (B,D) Bacterial growth curve (B) and corresponding area under growth (D) of phage SMacy. Bacterial growth was monitored for 8 hours at 30°C using a plate reader.
Figure 4. Inhibition and disruption of biofilm formation. (A,C) Quantification of biofilm formation by Raoultella planticola in the presence or absence of phage Macy (A) and Sally (C). Biofilms were stained with 0.1% crystal violet and quantified by measuring absorbance at 595 nm. (B,D) Percentage of biofilm disruption by phage Macy (B) and Sally (D) compared to untreated controls.
Burst size, latent period, and adsorption rate
To assess the replication dynamics and lytic potential of phages Sally and Macy, one-step growth assays were conducted. Macy exhibited a latent period of 20 min and a burst time of 25 min, producing an exceptionally high burst size with an average release of 8,375 virions per infected cell (Figure 5A). For Sally, the latent period was determined to be 20 min, with a burst time of 30 min and a burst size of 226 virions per infected RP8 cell, relatively lower than that observed for Macy (Figure 5B). Parallel adsorption assays revealed striking differences in binding efficiency, as nearly 50% of Sally particles adsorbed to host cells within 2 min of exposure (Figure 5C), compared to ~99% of Macy virions under the same conditions (Figure 5D). These results indicate moderate receptor-binding efficiency for Sally but exceptionally strong binding for Macy, whose rapid adsorption likely contributes to its large burst size and efficient infection dynamics. Although Macy exhibits a narrower host range and higher strain specificity, its rapid adsorption enables it to initiate infection effectively even at very low MOI.
Figure 5. Determination of infection dynamics of phage Macy and Sally. (A,B) One step growth curve showing the latent period, burst time and burst size of Macy (A) and Sally (B). (C,D) Adsorption curve of the phages Macy (C) and Sally (D).
Thermal and pH stability
The stability of Macy and Sally was evaluated under various temperatures and pH conditions to assess its their suitability for downstream applications. Thermal stability tests revealed that both Macy and Sally remained stable over a wide range of temperatures from 4°C to 45°C during a 24-hour incubation period (Figures 6A,B). At temperature higher than 55°C, a significant reduction in infectivity was detected in both phages. pH stability assays showed that Macy and Sally remained infective across a broad range of pH values (3–11), although both lost viability under highly acidic (<3) or strongly alkaline (>11) conditions (Figures 6C,D). This resilience highlights their potential utility in biocontrol applications under variable environmental conditions. This is particularly important for Sally, which infects clinical Raoultella strains, suggesting possible therapeutic relevance for gastric or other low-pH infections, where conventional antibiotics are less effective.
Figure 6. Effect of temperature and pH on the stability of the Raoultella planticola phage Macy and Sally. (A,B) Influence of temperature on viability of phage Macy (A) and Sally (B). Phage suspensions were incubated for 24 hours at different temperature. (C,D) Influence of pH on viability of phage Macy (C) and Sally (D). Phage suspensions were incubated for one hour at different pH regimes. Each experiment was conducted at least twice.
Genomic analysis
Comparative genomics revealed that phages Macy and Sally share only 55% nucleotide similarity (alignment data not shown), with scattered regions of homology, indicating distinct evolutionary origins. Phage Macy has a 147.8 kb genome (291 genes, 45% GC) with 77 bp direct terminal repeats characteristic of T4-like phages (Figure 7A and Supplementary Table S2). ICTV analysis classified Macy within Mydovirus (subfamily Vequintavirinae, family Myoviridae), most similar to Raoultella phage Ro1 (Figures 8A, 9A). Despite the relatedness to phage Ro1, Macy diverges substantially at the whole-genome level. A unique feature of Macy is an EPS depolymerase gene (locus tag MACY_240), absent in Ro1, consistent with its halo phenotype and potential role in biofilm disruption (Figure 9A and Supplementary Table S2). Macy and Ro1 share 204 homologous genes with 86.1% average amino acid identity, but exhibit a mosaic genome with at least 10 transcriptional units and 38 terminators, reflecting frequent recombination (Supplementary Figure S2 and Table 5). Phylogenomic analysis placed Macy in the Klebsiella II cluster, closely related to clinically relevant Klebsiella phages, and protein phylogenies revealed signatures of horizontal gene transfer with Escherichia, Pectobacterium, and Serratia phages (Figure 8A). Pangenome analysis further emphasized divergence between clusters: Macy and Ro1 shared 44% of genes but only 33–37% with Klebsiella phages, while Sally shared most core genes with Klebsiella/Enterobacter phages but retained the highest proportion of strain-specific genes (Supplementary Figure S2 and Table 5).
Figure 7. Genomic features of phages Macy and Sally. Circular genome map of phage Macy (A) and Sally (B). The genome of Macy is 147.8 kb in length with a GC content of 45% and the genome of Sally is 48.5 kb in length with a GC content of 56%. Open reading frames (ORFs) are color-coded based on predicted functional categories: DNA metabolism, structural proteins (head, tail, connector), lysis proteins, and hypothetical proteins.
Figure 8. Phylogenetic analysis of phages Macy and Sally. Phylogenetic tree generated using ViPTree based on whole-genome proteomic alignment of phage Macy (A) and Sally (B) with related members. Asterisks (*) indicate the positions of phages Macy (A) and Sally (B) within the trees.
Figure 9. Genome comparison between the Raoultella planticola phages and closely related Klebsiella and Enterobacter phages. (A) Comparative topology of the genomes of phages Macy and the closest relative Ro1. Percent amino acid identity represented by greyscale links, and each similarity group is assigned a unique color. (B) Synteny illustrating the degree of genomic conservation and gene order similarity of Sally and closely related phages. Open reading frames (ORFs) are color-coded based on predicted functional categories: DNA metabolism, structural proteins (head, tail, connector), lysis proteins, and hypothetical proteins.
Table 5. Comparative genomic features of Macy and closely related Raoultella and Klebsiella phages.
By contrast, Sally possesses a smaller 48.5 kb genome (66 genes, 56% GC), aligning more closely with its host genome (Figure 7B and Supplementary Table S3). It clustered within the genus Chivirus (family Casjensviridae), sharing 80.2% identity with Klebsiella phage VLCpiS8a, and was more closely related to Klebsiella than Raoultella phages (Figures 7B, 8B, 9B). Sally and VLCpiS8a share 21 homologous genes (Supplementary Figure S2). Pangenome analyses revealed a conserved genome organization with Klebsiella and Enterobacter phages, but Sally encoded the largest number of unique genes within its cluster, highlighting its distinct genetic profile (Figure 9B and Table 6). Together, these results demonstrate that Macy and Sally represent genetically distinct Raoultella phages: Macy is a large mosaic T4-like phage with signatures of horizontal gene transfer, while Sally is a compact Chivirus-like phage closely allied with Klebsiella/Enterobacter phages yet carrying unique genes.
Table 6. Comparative genomic features of Sally and closely related Klebsiella and Enterobacter phages.
Phage resistance and anti-phage defense mechanisms
To evaluate the fitness costs associated with phage resistance, spontaneous resistant mutants of R. planticola strain RP8 were generated through repeated exposure to high phage titers. Growth dynamics of these mutants were compared with the wild type by monitoring OD₆₀₀ over 16 h using a microplate reader. The Macy-resistant RP8 variant exhibited a measurable fitness cost, showing significantly higher bacterial densities when infected with phage Macy at an MOI of 100 compared with the wild-type strain under identical conditions (Figure 10A). A similar pattern was observed for the Sally-resistant RP8 variant (Figure 10B). However, while no significant difference was detected between the Macy-resistant and wild-type strains during the first 7 h of infection, after which the resistant mutant declined, the Sally-resistant RP8 maintained growth comparable to wild-type–infected cultures throughout the experiment. These results indicate that resistance to Sally imposed a greater physiological burden than resistance to Macy.
Figure 10. Identification of antiphage systems. Growth defects inImpaired growth of Macy (A) and Sally (B) resistant mutant of Raoultella planticola. (C) SNPs identified in the genome of Macy-resistant mutant. (D) SNPs identified in the genomes of Macy- and Sally-resistant mutants. and Sally-resistant mutant.
Whole-genome sequencing of the resistant mutants revealed single nucleotide polymorphisms (SNPs) linked to potential anti-phage defense mechanisms (Figures 10C,D). In the Macy-resistant mutant, a premature stop codon (Q795*) was identified in a glycosyltransferase gene responsible for synthesizing and modifying surface polysaccharides, likely altering receptor structure and preventing phage adsorption. Both Macy- and Sally-resistant mutants also shared a GATT motif substitution at position G3435100 in the promoter region of a LysR family glycine-cleavage-system transcriptional activator gene. This promoter mutation may affect LysR-mediated regulation, reshaping metabolic or stress-response pathways involved in phage defense. Together, these findings suggest that R. planticola acquires phage resistance through mutations that alter surface architecture and regulatory networks, conferring protection at the expense of growth fitness.
Discussion
Multidrug-resistant pathogens, particularly members of the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) represent the most formidable group of multidrug-resistant bacteria responsible for the majority of nosocomial infections worldwide (Dhungana et al., 2021). These organisms are known for their ability to “escape” the effects of existing antibiotics, prompting urgent calls for alternative therapeutic strategies. Although Raoultella species are not officially part of this group, their increasing isolation from clinical settings, particularly strains exhibiting carbapenem resistance such as R. planticola, suggests an emerging threat that mirrors the behavior of ESKAPE pathogens. The genetic and phenotypic similarity between Raoultella and Klebsiella, an established ESKAPE member, further underscores the potential for Raoultella to follow a similar trajectory in antimicrobial resistance and clinical relevance. As such, understanding and developing therapeutic options, including phage therapy, for drug-resistant Raoultella strains is a timely and important endeavor that aligns with global efforts to combat the growing challenge of antibiotic resistance.
In this study, we report the isolation and characterization of two virulent Raoultella phages, Macy and Sally, which display complementary features. Sally displayed a broad host range, infecting both environmental and clinical isolates of R. ornithinolytica and R. planticola, a characteristic shared with other cross-species Raoultella phages (Zamani et al., 2019; Kang et al., 2025; Fofanov et al., 2019). Such versatility may enhance therapeutic value in infections involving multiple strains. Macy, by contrast, was more specialized, infecting only R. planticola, but demonstrated exceptional replication parameters, including a remarkably high burst size (~8,375 PFU per cell) and very rapid adsorption (>99% within 2 min). This burst size is among the largest reported for bacteriophages, surpassed only by Hafnia phage Ca (Pan et al., 2022). The combination of high replication and rapid adsorption suggests Macy can efficiently suppress bacterial populations while reducing opportunities for resistance emergence.
An additional therapeutic feature of Macy is its encoded EPS depolymerase, consistent with the halo formation observed on host lawns. EPS depolymerases degrade extracellular polysaccharides in capsules or biofilms, facilitating phage access to bacterial cells and enhancing infection efficiency in both planktonic and biofilm-associated states (Chen et al., 2022; Vukotic et al., 2020; Wang et al., 2020). This activity is particularly valuable against Raoultella biofilms, which exhibit increased antibiotic tolerance and persistence in clinical and environmental settings (Hall-Stoodley et al., 2004; Hoiby et al., 2010; Donlan, 2000; Andrés-Lasheras et al., 2024). By breaking down the polysaccharide matrix, Macy can penetrate deeper biofilm layers and lyse otherwise protected bacterial cells, supporting its potential as a potent therapeutic agent. Although Sally lacks an identifiable depolymerase, its broader host range provides a complementary advantage for potential treatment applications.
Genomic analyses highlighted distinct evolutionary trajectories. Macy, a 148-kb dsDNA phage with ~45% GC content, displays a mosaic genome typical of T4-like viruses, reflecting frequent recombination and modular exchange (Grose and Casjens, 2014). Phylogenetically, it clustered within the Klebsiella II clade alongside phage Ro1, indicating shared ancestry with clinically relevant phages (Adriaenssens and Brister, 2017). The conservation of its lysis cassette, including i-spanin, o-spanin, and endolysin underscores strong purifying selection on structural and lytic modules (Cahill and Young, 2019; Young, 2013). In contrast, Sally has a smaller 48.5 kb genome (56% GC), aligning closely with its host genome and suggesting tighter co-adaptation. As a Chivirus (family Casjensviridae), it is more related to Klebsiella phages than other Raoultella phages. Despite overall genomic conservation, Sally encodes the largest number of unique genes in its proteomic cluster, highlighting its evolutionary distinctiveness.
Pangenome analysis further revealed divergent strategies, with the Raoultella phages (Macy and Ro1) encoding ~68% more strain-specific genes than related Klebsiella phages, suggesting stronger host-driven specialization. Only 27% of genes were shared across all examined genomes, consistent with high genomic plasticity in T4-like phages. By contrast, Sally and related Chivirus phages showed smaller genomes, higher conservation, and fewer strain-specific genes. These differences point to Raoultella phages as an underexplored but diverse evolutionary group.
Finally, mutations conferring resistance to Macy and Sally were primarily associated with alterations in surface glycosyltransferase genes and regulatory elements, consistent with mechanisms reported in other Gram-negative bacteria where receptor modification serves as a dominant antiphage strategy (Washizaki et al., 2016). The identified LysR-promoter mutation suggests that transcriptional reprogramming may complement structural receptor changes, reflecting a multi-layered defense system analogous to metabolically driven antiphage responses observed in Pseudomonas and Escherichia coli (Liang et al., 2025; Fitzpatrick et al., 2025). Although such adaptations effectively block phage infection, they often impose measurable fitness costs, reinforcing the evolutionary trade-offs that maintain phage–host coexistence in natural environments (Liang et al., 2025).
A key limitation of this study is the absence of in vivo validation, which is critical to assess therapeutic efficacy, stability, and ecological interactions under physiological conditions. Ongoing efforts, including evolutionary phage training, in vivo infection modeling, and phage pharmacokinetic studies, aim to address this gap and provide the necessary evidence for translational development.
Conclusion
This study identifies two novel bacteriophages, Macy and Sally, as promising candidates for phage therapy against multidrug-resistant R. planticola. Their complementary biological properties, including Macy’s rapid replication and depolymerase activity and Sally’s broad host range and strong anti-biofilm capabilities, highlight their therapeutic potential. Together with genomic insights into phage resistance mechanisms, these findings advance the foundational knowledge required to develop targeted, evolution-informed phage cocktails. Future studies will expand therapeutic evaluations across additional clinical isolates and progress toward translational applications, ultimately supporting the development of effective alternative treatments for carbapenem-resistant Raoultella infections.
Data availability statement
The complete genome sequences of Macy and Sally are available in GenBank under accession numbers PX290106 and PX290107, respectively.
Author contributions
CH: Writing – original draft, Investigation, Visualization, Formal analysis, Resources, Supervision, Project administration, Validation, Writing – review & editing, Methodology, Data curation. JF: Writing – review & editing, Data curation, Methodology. LB: Methodology, Writing – review & editing, Data curation. AMu: Data curation, Methodology, Writing – review & editing. VL: Writing – review & editing, Data curation, Methodology. DN: Methodology, Writing – review & editing, Data curation. SG: Writing – review & editing, Methodology, Data curation. AMa: Resources, Writing – review & editing, Methodology. CM: Data curation, Visualization, Supervision, Validation, Writing – review & editing, Methodology, Resources. YD: Writing – review & editing, Data curation, Validation, Supervision, Resources, Visualization, Methodology. OO: Visualization, Resources, Conceptualization, Validation, Project administration, Funding acquisition, Investigation, Methodology, Supervision, Formal analysis, Writing – original draft, Data curation, Writing – review & editing.
Funding
The author(s) declare that financial support was received for the research and/or publication of this article. This study was supported by initial complement package provided to OO by the College of Natural and Agricultural Sciences, University of California, Riverside.
Acknowledgments
The authors would like to express their gratitude to Ryan Quaal and Hajun Lee for a critical review of this manuscript.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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The authors declare that no Gen AI was used in the creation of this manuscript.
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Supplementary material
The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2025.1726803/full#supplementary-material
Footnotes
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Keywords: carbapenem-resistant Raoultella planticola, bacteriophage therapy, host range, clinical strains, multidrug-resistant pathogens
Citation: Hoang CV, Fan J, Bhasin L, Del Mundo A, Law V, Nguyen D, Ganiger S, Mansour A, McElheny CL, Doi Y and Olawole OI (2026) Two virulent bacteriophages targeting carbapenem-resistant Raoultella planticola. Front. Microbiol. 16:1726803. doi: 10.3389/fmicb.2025.1726803
Edited by:
Karen Fong, Agriculture and Agri-Food Canada (AAFC), CanadaReviewed by:
Gunaraj Dhungana, Meharry Medical College, United StatesThomas Brenner, Ministry of Agriculture, Food and Rural Affairs, Canada
Copyright © 2026 Hoang, Fan, Bhasin, Del Mundo, Law, Nguyen, Ganiger, Mansour, McElheny, Doi and Olawole. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Olakunle I. Olawole, b2xha3VubGUub2xhd29sZUB1Y3IuZWR1