Genomic and translational characterisation of Autographviridae phage SAKp26.2 for catheter-associated biofilm clearance in drug-resistant Klebsiella pneumoniae

Genomic and translational characterisation of Autographviridae phage SAKp26.2 for catheter-associated biofilm clearance in drug-resistant Klebsiella pneumoniae | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genomic and translational characterisation of Autographviridae phage SAKp26.2 for catheter-associated biofilm clearance in drug-resistant Klebsiella pneumoniae Aafreen ., Sambuddha Chakraborty, Deepangkar Chisim Sangma, Anusha Jatley, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9111241/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 19 You are reading this latest preprint version Abstract AMR has become a multi-million death crisis primarily driven by carbapenem-resistant Gram-negative infections, and it is projected to worsen sharply by 2050, especially in South Asia, unless major interventions are implemented. One of the major contributors to this burden is Klebsiella pneumonia. The WHO has categorised carbapenem-resistant Klebsiella pneumoniae as a critical priority pathogen, and the current prevalence of carbapenem-resistant hypervirulent K. pneumoniae has raised concerns about potential future pandemics. The bacteria can mediate the transfer of resistance from environmental to clinical strains. Moreover, biofilm-associated infections caused by multidrug-resistant K. pneumoniae exacerbate healthcare challenges, particularly in hospital-acquired infections associated with medical devices such as catheters and implants. Amid escalating antibiotic failures, bacteriophages have re-emerged from the pre-antibiotic era as a new-age therapeutic alternative to combat drug-resistant bacterial infections. We report SAKp26.2, a novel Autographviridae phage isolated from hospital sewage, which acts as a potent antibacterial and antibiofilm agent against several clinical, drug-resistant K. pneumoniae strains. Combination treatment of SAKp26.2 with antibiotics resulted in a significant delay in the emergence of treatment resistance compared to monotherapy, supporting its potential as a phage-antibiotic synergistic therapeutic. The phage has a genome size of 41,282 bp and lacks any virulence or antibiotic resistance genes. SAKp26.2 is a strong depolymerase-producing phage and is equipped with other critical lysis-associated enzymes. A rapid elimination of biomass and bacteria residing in biofilms was achieved, resulting in a 99% reduction within 4 hours. Additionally, our study illustrates an association between efficiency of plating and kill-kinetics performance, reflecting how phage replication efficiency within a host population may influence the epidemiological spread of infection. Notably, the phage also showed significant biofilm clearance from urinary tract catheters, indicating potential biomedical applications. Overall, this study integrates fundamental phage biology with a clinically relevant scenario, bridging the gap between bench and bedside. Klebsiella pneumoniae Anti-microbial resistance bacteriophage bacterial-biofilm phage-antibiotic combination efficiency of plating Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Antimicrobial resistance (AMR) has emerged in the current century as one of the major contributors to public health challenges. The rapid spread of resistance renders conventional antibiotic treatment ineffective for adult healthy individuals but severely limits its effectiveness for most hospital-acquired bacterial infections in immunocompromised patients. Realising the urgency, the World Health Organization (WHO) has highlighted the necessity for an improvement and coordinated global effort to contain AMR especially for ESKAPE pathogens [ 1 ]. Klebsiella pneumoniae is one of the ESKAPE group pathogens that acquires antibiotic resistance at an alarming rate. In 1929, resistance to beta-lactam antibiotics in gram-negative was discovered by Alexander Fleming. Since then, extensive studies have been done about K. pneumoniae and was found that it produces a beta-lactamase that causes hydrolysis of beta-lactam ring in antibiotics hence resulting in an intrinsic resistance to cell wall synthesis targeting antibiotics like ampicillin and first-generation penicillin [ 2 ]. In 1983 and 1989 cases of Extended-spectrum beta-lactamase (ESBL) K. pneumoniae were reported. ESBLs can hydrolyse oxyimino cephalosporins significantly reducing the efficiency of treatments with third-generation cephalosporins. This led to carbapenems as an effective antibiotic against ESBLs [ 2 ]. In course of time negligent application and disposal of antibiotic made bacteria adapt to carbapenems as well. K. pneumoniae uses human as their primary reservoir, mainly colonizing in patient’s gastrointestinal tract and the hands of hospital personals. Higher numbers of such cases have been reported in South Asia [ 3 ][ 4 ] and more importantly the mortality rate of patients with pneumonia caused by K. pneumoniae is about 50% [ 5 ]. K. pneumoniae passes on to their next generation the antimicrobial resistant genes through mobile elements such as plasmids, transposons and integrons. These elements play critical role in vertical and horizontal transmission of the genes [ 6 ]. These might confer advantage against antibiotic by producing specified enzymes [ 7 ], decreased cell permeability by cutting-off Omps [ 8 ], overexpression of efflux pumps such as KpnGH [ 9 ] and modifying the key target of the antimicrobial agent [ 10 ]. Along with these features some K. pneumoniae is capable of biofilm formation further exacerbating its clinical impact [ 11 ]. Infections associated with biofilms are extremely difficult to deal with. The resistant K. pneumoniae biofilms manage to survive in healthcare environments and on the devices used for treatment enhancing their infectivity [ 12 ]. One such matter is the formation of biofilm by K. pneumoniae on the catheter of patients with urinary tract infection (UTI). Each day a catheter remains indwelled in a patient, the risk of biofilm formation on the wall of catheter increases 3.55 times, and with 3 days the chances of UTI become 90% [ 13 , 14 ]. The effective way to counter such is to daily evaluation of patient for early catheter removal but many patients with UTIs associated with catheters cannot have their removed due to their underlying disease conditions [ 15 ]. With such limitations on patients along with rapid increase in the acquired resistance and failure of conventional antibiotics, the development of novel antimicrobial strategies is what humanity requires. To limit the impact of AMR is of utmost importance, as the present figure of cases of antimicrobial-resistant-bacterial infections indicates arrival of a pandemic [ 16 ]. Novel countermeasures are required to be developed against AMR since, it is an unavoidable phenomenon, which renders the drug’s effectiveness [ 17 ]. An alternate to antibiotics might keep the AMR in-check, as inappropriate usage and exposure of antibiotic is resulted in huge disease burden and accelerated emergence of AMR, and has shorten the list of effective antibiotics. In face of this challenge, bacteriophages have re-emerged as promising therapeutic candidates. Phage therapy, which predates the antibiotic era, offers several advantages, including high specificity toward bacterial hosts [ 18 ], self-amplification at the site of infection [ 19 ], and the ability to target antibiotic-resistant bacteria [ 20 ]. Importantly, certain bacteriophages encode depolymerases and lytic enzymes capable of degrading bacterial capsules and biofilm matrices [ 21 ], making them particularly preferrable for treating biofilm-associated infections. Furthermore, combining bacteriophages with antibiotics has shown potential to enhance antibacterial efficacy [ 22 ] and delay the emergence of resistance, suggesting a synergistic therapeutic approach [ 23 ]. In this study, we report the isolation and characterization of SAKp26.2, a novel lytic bacteriophage belonging to the family Autographviridae , isolated from hospital sewage. We evaluated its antibacterial and antibiofilm activity against a panel of clinically relevant, drug-resistant K. pneumoniae strains. In addition to genomic characterization, we investigated its biofilm clearance capacity, and efficiency, and highlighted the correlation between efficiency of plating and kill kinetics which shows a very interesting result that can be mirrored in clinical setups. With clinically relevant infection catheter-associated biofilms, this work aims to advance the translational potential of phage-based therapeutics and contribute to the development of effective strategies against multidrug-resistant K. pneumoniae . 2. Material and Methods 2.1 Bacterial Strains and Growth Conditions The multi drug-resistant (MDR) K. pneumoniae strains were acquired from Madras Medical Mission Hospital (MMMH) in Chennai, India, were cultured at 37 ºC in Luria-Bertani Broth with shaking at 150 rpm, unless otherwise stated. Viable cell counts were estimated by spotting serial dilutions of the culture onto LB agar plates and incubating at 37°C overnight. The number of colony-forming units per mL was determined. All experiments were conducted in triplicate. 2.2 Isolation and Purification of Bacteriophage The bacteriophage SAKp26.2 was isolated from sewage water collected at the Madras Medical Mission Hospital (MMMH), Chennai, India, following the protocol described by Chakraborty et al. [ 24 ]. Briefly, 5 mL of mid-log-phase XDR K. pneumoniae clinical isolate U5877, grown to an OD 600 of 0.3–0.6, was added to 5 mL of sterile LB medium. The mixture was incubated overnight at 37°C with shaking at 150 rpm, then centrifuged at 12,000× g for 10 min at room temperature. The presence of lytic bacteriophages in the supernatant was preliminarily confirmed by a spot test, in which 10 µL of the supernatant was spotted onto the top of the lawn of the host bacterial isolate and allowed to dry. The plate was incubated at 37 ºC overnight. A clear zone of lysis suggests positive bacteriolytic activity. A single phage was purified using a double agar overlay technique. The supernatant was serially diluted tenfold. 3 mL of 0.5% soft agar containing 100 µL of diluted supernatant and 500 µL of an overnight culture of K. pneumoniae was poured onto a 1.5% LB agar plate and incubated overnight at 37°C. The next day, a single plaque was picked, inoculated into an early-log-phase culture, and incubated overnight. The suspension was centrifuged at 12,000× g for 10 min at room temperature, and the supernatant was used for a double-layer agar overlay experiment. This procedure was repeated three times to get a single purified phage. Finally, the supernatant was treated with chloroform in a 1:10 ratio for 15 minutes. After chloroform treatment, the suspension was centrifuged, filtered through a 0.22 µm syringe-driven filter, and stored at 4°C. 2.3 Whole genome sequencing and bioinformatic analysis of SAKp26.2 The phage suspension was treated with DNase (1 µg/mL) and RNase (1 µg/mL) for 1 h to eliminate any extracellular bacterial DNA and RNA. The mixture was subsequently heated at 80°C for 15 min to inactivate the enzymes. After enzyme inactivation, 50 µg/mL proteinase K and 5% SDS were added to the phage suspension and incubated for 1 h at 56°C to denature the phage capsid proteins. The suspension was then mixed with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) and centrifuged at 13,000 × g for 5 min at 4°C. The aqueous supernatant was carefully collected and mixed with an equal volume of chilled isopropanol, followed by incubation at − 20°C for 1 h to precipitate the phage DNA. The mixture was centrifuged to pellet the DNA, and the resulting pellet was washed with 1 mL of 75% ethanol and centrifuged at 12,000 × g for 10 min at 4°C. The DNA pellet was then air-dried, resuspended in 30 µL of ddH₂O, and stored at − 20°C until further use. The concentration and purity of the extracted DNA were assessed using a Qubit 4.0 Fluorometer, and DNA integrity was verified on a 0.8% agarose gel prepared in 1× TAE buffer. Whole-genome sequencing of phage SAKp26.2 was performed using the Illumina HiSeq platform. Raw sequencing reads were subjected to quality control using Fastp (version 0.20.1), a multithreaded C + + based tool. De novo genome assembly was performed using the St. Petersburg genome assembler (SPAdes) ( https://bio.tools/spades ). The phage sequence was extracted using PHASTER. Genome annotation was initially carried out using Prokka (version 1.14.6) ( https://proksee.ca/tools/prokka ), followed by further functional annotation using additional tools including the RAST server version 2 ( http://RAST.nmpdr.org ), BLASTp ( https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins ), InterPro ( https://www.ebi.ac.uk/interpro/ ), UniProt ( https://www.uniprot.org/ ), and GenomeVx ( http://conantlab.org/GenomeVx/ ) for genome visualization. The GC content of the phage genome was calculated and visualized using GC Content Plotter. The presence of tRNA genes in the genome was assessed using tRNAscan-SE version 2.0 ( http://trna.ucsc.edu/tRNAscan-SE/ ). Phylogenetic analysis was performed using MEGA11 software based on both the terminase large subunit sequence and the whole genome sequence. Comparative genomic analysis with related phages was conducted using DiGAlign ( https://www.genome.jp/digalign/upload ).. Since the therapeutic application of phages requires the absence of virulence factors and antibiotic resistance genes, the genome was screened against the Virulence Factor Database (VFDB) ( https://www.mgc.ac.cn/VFs/main.htm ) to detect virulence-associated genes and against ResFinder 4.1 ( https://cge.food.dtu.dk/services/ResFinder/ ) to identify potential antimicrobial resistance determinants 2.4 Transmission Electron Microscopy 10 µL of Poly Ethylene Glycol (PEG)-precipitated phage suspension (~ 10¹¹ PFU/mL) was placed onto a clean Parafilm surface. A 300-mesh carbon-coated copper grid (EMS200-Cu; Electron Microscopy Sciences, USA) was gently positioned over the droplet and allowed to adsorb the sample for 25–30 min. The grid was then carefully lifted, and excess fluid was removed by gentle blotting with filter paper. For negative staining, the grid was inverted (sample side down) onto a drop of 2% (w/v) aqueous phosphotungstic acid solution (pH 6.8) and incubated for 3 min. Residual stain was blotted off, and the grid was air-dried on filter paper to prevent contact with any wet surface. Once completely dried, the grids were visualised and imaged using a Thermo Scientific™ Talos L120C transmission electron microscope (operating at 20–120 kV) at the All-India Institute of Medical Sciences (AIIMS), New Delhi, India. 2.5 Host Adsorption Assay Host adsorption assay was performed according to the procedure described by Mizuno et al. with slight modifications [ 25 ]. Briefly, SAKp26.2 phage particles were mixed with 26 clinical isolates of K. pneumoniae with a multiplicity of Infection (MOI) of 0.1 in different tubes and incubated at 37 ºC in static conditions for 40 minutes. At 5-min intervals, microcentrifuge tubes were removed, centrifuged for 4 min at 12,000x g, and the supernatant was collected to determine unabsorbed phages using the small-drop plaque assay. 2.6 Determination of Phage Life Cycle by One-Step Growth Curve A one-step growth curve to determine the latency and burst size of the phage was performed as described by Amarillas et al., with slight modifications [ 26 ]. 26 clinical isolates of K. pneumoniae were grown to a mid-exponential phase (OD: 0.6). Then, 0.9 ml of mid-log phase was mixed with 0.1 ml of SAKp26.2 in tube-A, MOI 0.1, and kept for 10 min (according to previously evaluated adsorption time) at 37°C in static condition. After incubation, 0.1 ml of the phage–bacteria suspension was mixed with 9.9 ml of phosphate-buffered saline (PBS) in tube B, and the mixture was vortexed several times. Then 0.1ml from tube B was aspirated and dispensed into tube C, containing 9.9 ml of Luria-Bertani broth, to prepare the mixture. Tube-C was kept in a static incubator at 37 ºC for 140 min. 0.1 ml of the bacterial-phage suspension was taken at 10 min intervals. At each time interval, the sample taken out was centrifuged at 12,000× g for 3 min, and the number of unadsorbed bacteriophage in the supernatant was determined by titration. 2.7 Assessment of Phage Stability pH, thermal and solvent stability of the SAKp26.2 were assessed according to the protocol described by Chauhan et al, 2024 [ 21 ]. 2.7.1 pH stability - The pH of LB broth was adjusted to values ranging from 1 to 14 (pH 1, 3, 5, 7, 8, 9, 10, 12, and 14) using 0.1 N HCl or 1 N NaOH. The phage suspension was then mixed with the pH-adjusted media at a 1:1 ratio to achieve a final concentration of approximately 1 × 10⁹ PFU/mL. The mixtures were incubated at 37°C for 1 h, after which phage titers were determined to evaluate stability under varying pH conditions. 2.7.2 Thermal stability - Phage suspensions (~ 1 × 10⁹ PFU/mL in PBS) were exposed to different temperatures (37°C, 55°C, 65°C, 75°C, 85°C, and 100°C) for 1 h. After incubation, residual phage infectivity was determined by plaque assay. 2.7.3 Solvent stability - Phage stability in different solvents was evaluated by mixing equal volumes (1:1) of phage suspension with phosphate-buffered saline (PBS), chloroform, or 0.9% saline, resulting in a final concentration of around 1 × 10⁹ PFU/mL. The mixtures were incubated at 37°C for 1 h with shaking at 150 rpm. Following incubation, samples were centrifuged at 12,000 × g for 10 min, and the supernatant was collected for phage titer determination. 2.8 Spot Test Assay: The spot test assay was performed according to the procedure described by Chakraborty et al. (2024) with brief modifications [ 24 ]. The PEG-concentrated phage suspension was serially diluted from 10⁹ PFU/mL to 10 PFU/mL, corresponding to 10⁻¹ to 10⁻¹⁰ dilutions. Subsequently, 10 µL of each dilution was spotted onto a lawn of mid-log phase bacterial culture prepared on agar plates. The plates were then incubated at 37°C overnight, and lytic activity was assessed based on the formation of clear zones. 2.9 Efficiency of Plating (EOP) EOP was determined according to the procedure described in Mirzaei et al, 2015 [ 27 ] with slight modifications. Based on observations of host range of phage SAKp26.2, 16 clinical isolates of K. pneumoniae ; O3933, P4272, U4317, U4444, U4636, P4725, U5485, B5521, U5877, U6350, U6795, U6875, U7224, U198, O775, and U2150; were selected as they showed a range of susceptibility to SAKp26.2 infection. Overnight cultures of 16 clinical isolates of K. pneumoniae were prepared in LB broth. Phage suspensions were serially diluted from stock in SM buffer. Each diluted phage suspension was mixed with the corresponding overnight bacterial culture at a ratio of 1:9 (phage: bacteria) resulting in decreasing MOI. The mixture was incubated statically at 37°C for 10 min to allow phage adsorption. Post incubation 20 µL of each phage-bacterial mixture was spotted onto LB agar plates and allowed to air dry. The plates were then closed, inverted, and incubated overnight at 37°C. Plaques formed on the bacterial lawns were examined to assess the degrees of phage infectivity across the clinical isolates. The number of plaques obtained for each isolate was compared with those formed on the reference host strain U5877, which was used for phage amplification. The assay was performed in triplicate to ensure reproducibility. To check corelation between EOP and kill kinetics; overnight cultures of the clinical isolates were co-incubated with bacteriophage SAKp26.2 at a multiplicity of infection (MOI) of 1 in individual wells of a sterile 96-well flat-bottom microtiter plate. The plate was incubated at 37°C for 24 hours in an BioTek Epoch-2 microplate reader (Agilent Technologies, USA), with optical density (OD) measurements recorded at 600 nm at defined time intervals. Each assay was performed in triplicate. For each clinical isolate, a corresponding control well containing bacterial culture without phage was included to serve as a growth control. 2.10 Analysis of Antibacterial Efficacy of the Bacteriophage SAKp26.2 A kill-kinetics study was conducted to evaluate the lytic activity of the phage against its host, K. pneumoniae clinical isolate U5877. The bacterial culture was grown to the mid-exponential phase and harvested by centrifugation at 6,000 × g for 5 minutes. The supernatant was discarded, and the pellet was resuspended in fresh growth medium. The resulting suspension was then aliquoted into microcentrifuge tubes. The bacteriophage was added to the bacterial suspensions at multiplicities of infection (MOIs) of 0.1, 1, 10, and 100. Subsequently, 200 µL of each phage–bacteria mixture was transferred in triplicate into a 96-well microtiter plate. Bacterial growth kinetics were monitored for 5 hours using a BioTek Epoch-2 microplate reader (Agilent Technologies, USA), with optical density at 600 nm recorded every 20 minutes. 2.11 Assessment of antibiofilm potential of SAKp26.2 and biofilm fluorescent staining In-vitro antibiofilm assays were initially performed in microtiter plates following the method described by Chauhan et al, 2012 [ 28 ] with slight modifications. Overnight cultures of host, K. pneumoniae clinical isolate U5877, were diluted 1:100 in fresh LB broth and incubated statically at 37°C for 24 h to allow mature biofilm formation. After incubation, planktonic cells were removed, and wells were washed three times with 1X PBS. Phage suspension (200 µL; MOI 1) was added to the established biofilms and incubated at 37°C under static conditions for 4, 8, and 12 h. Control wells treated with 1X PBS. Following treatment, wells were washed thrice with PBS. Biofilm reduction was evaluated by viable cell counts and crystal violet (CV) staining. For CFU determination, biofilms were scraped, resuspended in PBS, serially diluted, and plated on LB agar plates, which were incubated at 37°C. For biofilm biomass quantification, wells were stained with 0.1% CV, incubated at room temperature for 15 min, rinsed with 1X PBS three times, and allowed to dry. The bound dye was resuspended in destaining solution [acetone: ethanol (1:4)] and transferred into a fresh 96-well plate. Absorbance was measured at 595 nm. The experiment was performed in triplicate. For biofilm fluorescent staining, a mixture of dyes consisting of SYTO 9 (30 µM final concentration) and propidium iodide (5 µM final concentration) in a 1:6 ratio was prepared in autoclaved distilled water by combining 1.5 µL of each dye stock with 997 µL distilled water. Finally, approximately 50 µL of this dye mixture was added to each well to cover the well surface evenly, and the wells were incubated at room temperature in the dark for 15 minutes. Following incubation, unbound dye was removed, and the wells were washed three times with 1X PBS. Biofilms were observed using an inverted fluorescence microscope (EVOS M5000, ThermoFisher, Massachusetts, USA), in which SYTO 9 stained the cells green, and PI stained the dead cells red. All procedures were carried out under minimal light exposure to avoid dye photobleaching. post-treatment and washing, 2.12 Phage-Antibiotic Combination Treatment-Mediated Modulation of Bacterial Growth Kinetics A broth microdilution assay was conducted to evaluate the growth kinetics of MDR K. pneumoniae clinical isolate U5877 under treatment with varying antibiotic concentrations, different phage multiplicities of infection (MOIs), and phage–antibiotic combinations. Briefly, 100 µL of LB broth was dispensed into each well of a 96-well microtiter plate. Four experimental conditions were included: broth control, antibiotic alone, phage alone, and antibiotic–phage combination. Gentamicin was two-fold serially diluted in LB broth, ranging from 1.25 mg/mL to 0.61 µg/mL. Phage suspensions were similarly two-fold serially diluted to achieve MOIs ranging from 100 to 0.048. An equal volume of exponentially growing bacterial culture was added to each well to obtain a final inoculum of 5 × 10⁵ CFU/mL. Bacterial growth was monitored for 24 h using a BioTek Epoch-2 microplate reader (Agilent Technologies, USA), with optical density measured at 600 nm at 1-h intervals. The same experimental procedure was performed using ceftazidime in place of gentamicin. 2.13 In-vitro antibiofilm activity of SAKp26.2 in urinary tract catheter using continuous flow set up In-vitro efficacy of phage SAKp26.2 against device-associated biofilms of MDR K. pneumoniae isolate was evaluated using a previously described continuous flow system [ 28 ]. Approximately 10² bacterial cells in 100 µL were injected through the silicon septum of a totally implantable venous access port (TIVAP) catheter. The bacterial cells were allowed to adhere to the internal surface of the TIVAP for 3 h at 37°C. Following adhesion, the continuous flow system was initiated at a flow rate of 300 µl/min and maintained for 24 h to allow biofilm formation within the catheter lumen. Non-adherent cells and spent media were continuously removed and collected in a waste container. After biofilm development, the TIVAP-associated biofilms were treated with SAKp26.2 for 4 h and 8 h under static conditions by instilling the phage suspension into the catheter lumen. Following treatment, biofilm cells were recovered and viable counts were determined by CFU/mL plating. Untreated TIVAP-associated biofilms served as controls. 2.14 Scanning electron microscopy of the urinary tract catheters For visualization of biofilm architecture and phage-mediated disruption, field emission scanning electron microscopy (FESEM) was performed with slight modifications of previously described method [ 28 ]. After aseptic removal of colonized totally implantable venous access ports (TIVAP) from the flow system, approximately 1 cm segments from the catheter tip were cut, and the septum was carefully dissected from the port using a sterile scalpel. The septum and catheter segments were washed twice with sterile PBS buffer to remove loosely attached debris and subsequently the catheter segments were fixed overnight in 2.5% glutaraldehyde prepared in PBS at 4°C. Following fixation, the samples were washed with PBS and subjected to sequential dehydration using graded ethanol concentrations (10, 20, 30, 40, 50, 60, 70, 80, 90, and 100%) for 10 min each. The dehydrated samples were air-dried and sputter-coated with gold prior to FESEM imaging at USIC University of Delhi south campus with ZEISS Gemini SEM 500. 2.15 Statistical analysis All experiments in this study were performed with a minimum of three independent biological replicates to ensure reproducibility. Statistical analyses were carried out using GraphPad Prism (version 10.0). Differences between groups were evaluated using an unpaired t-test or one-way analysis of variance (ANOVA), as appropriate. A p -value of less than 0.05 was considered statistically significant. 3. Results 3.1 SAKp26.2 exhibits rapid adsorption, short latent period, high burst size, and stability under physiological conditions The bacteriophage SAKp26.2 was isolated from the sewage water sample collected at Madras Medical Mission Hospital, from where Klebsiella pneumoniae clinical strains were also isolated. The phage formed characteristic bullseye plaques on the lawn of the host K. pneumoniae U5877. The plaques exhibited a clear, core lysis zone measuring approximately 0.20 cm in diameter, surrounded by a semi-transparent halo ( Fig. 1 a ). The presence of a halo suggests depolymerase activity resulting from enzymatic degradation of capsular polysaccharides on the bacterial surface [ 21 ]. TEM analysis revealed that the phage SAKp26.2 possessed an icosahedral head and a short, non-contractile tail, structural features characteristic of members of the family Autographviridae [ 29 ]. The capsid diameter was approximately 50 nm ( Fig. 1 b ). TEM images show the adsorption of SAKp26.2 on host bacterial cells, as well as their lysis (Fig. S1 -S3). Phage SAKp26.2 exhibited rapid adsorption to its host strain with nearly 95% of phage particles adsorbed within 5 minutes ( Fig. 1 c ). The one-step growth curve indicated a short latent period of around 10 minutes, followed by a rise period of 50 minutes. A secondary latent phase (plateau) was observed thereafter. The calculated burst size was approximately 166 PFU per infected cell, indicating the release of 166 progeny virions upon lysis of a single bacterial cell ( Fig. 1 d ). The stability of the phage SAKp26.2 was evaluated under varying pH, temperature, and solvent conditions. A marked reduction in phage titer was observed at extreme acidic and alkaline pH. The phage remained stable at neutral pH, followed by a sharp decline at pH 12, and complete degradation of virion particles occurred at pH 1, 3 and 14 ( Fig. 1 e ). Thermal stability analysis showed that SAKp26.2 retained infectivity up to 55 ºC. However, a sharp decline in phage titer was recorded at 65 ºC and 85 ºC, followed by complete inactivation at 100 ºC ( Fig. 1 f ). Solvent stability assessment indicated that SAKp26.2 remained stable in 0.9% saline and PBS ( Fig. 1 g ). 3.2 SAKp26.2 possesses a ~ 41 kb modular lytic genome and a novel species within Autographviridae Phage SAKp26.2 genomic DNA was successfully isolated as a single high-molecular-weight band (~ 41 kb) on agarose gel electrophoresis (Fig. S4) . Whole-genome sequencing using the Illumina platform generated a 41,467 bp linear double-stranded DNA genome (GenBank accession: PX974311) with 97% assembly coverage and a GC content of 52.99% (Fig. S5) . A total of 50 coding sequences (CDSs) were predicted (supplementary table 2) , and no tRNA genes were identified. Genome annotation using Prokka and RAST, followed by manual validation (BLASTp, InterPro, UniProt), revealed a modular organization typical of the family Autographiviridae. The genome is arranged unidirectionally and comprises distinct functional modules including DNA packaging (terminase small and large subunits), host replication machinery takeover proteins (S-adenosyl-L-methionine hydrolase, serine/threonine kinase, and phage-encoded RNA polymerase), DNA replication and nucleotide metabolism (DNA polymerase, primase/helicase, exonuclease, ligase, HNH endonuclease), structural assembly (major capsid protein, scaffolding protein, head-to-tail connector, tail tubular proteins, and tail fiber), DNA injection/ejectosome components, and a complete lysis cassette (endolysin, class II holins, and Rz-like protein) ( Fig. 2 a ) . The presence of a single-subunit DNA-dependent RNA polymerase confirms its classification among T7-like Autographiviridae phages [ 30 ]. Notably, CDS-4 encodes a putative S-adenosyl-L-methionine hydrolase and CDS-5 a serine/threonine kinase, while CDS-26 was flagged by DefenseFinder as a potential counter-anti-phage defense protein, suggesting adaptation to overcome host restriction or intracellular immunity systems. CDS-47 encodes a DNA ejectosome-associated peptidoglycan lytic exotransglycosylase, indicating an enzymatically facilitated DNA translocation mechanism. Approximately one-third of CDSs were annotated as hypothetical proteins, representing potential strain-specific adaptive elements (supplementary table 2) . Lifestyle prediction of SAKp26.2 has marked it as a virulent phage (Fig. S6) , and comprehensive screening revealed absence of integrase, repressor, excisionase, antibiotic resistance genes, virulence factors, and canonical lysogeny modules, supporting a strictly lytic lifestyle and genomic safety (supplementary table 2 & Fig. 2 a ) . Phylogenetic analysis based on whole-proteome comparison and terminase large subunit clustering placed SAKp26.2 within the family Autographiviridae . Whole-genome phylogenetic analysis identified Klebsiella phage RSU-F9L as the closest relative ( Fig. 2 b ) . Whole-genome comparison revealed 93.30%, nucleotide similarity with Klebsiella phage RSU-F9L and an average nucleotide identity (ANI) value of 91.35% (Fig. S7) . Phylogenetic reconstruction based on the terminase large subunit with the most closely related 20 phages identified Klebsiella phage PEA128 as the closest relative in the TerL-based tree ( Fig. 2 c ) . ANI analysis with Klebsiella phage PEA128 showed 91.35% identity, which remains below the 95% species demarcation threshold defined by ICTV (Fig. S8) . These results collectively confirm that SAKp26.2 represents a novel species within the Autographiviridae lineage. VIRIDIC intergenomic similarity and Protein Clusters (PC) based intergenomic distance analyses consistently demonstrated values below the 95% ICTV species demarcation threshold while remaining above the 70% genus cutoff ( Fig. 2 d & Fig. S9 ). SAKp26.2 (41,467 bp) showed strong similarity with the RSU-F9L genome (~ 39.4 kb) while maintaining distinct regions of sequence divergence. These differences likely correspond to strain-specific genes or hypothetical proteins that may contribute to host adaptation or functional variation. Overall, the alignment supports the close evolutionary relationship of SAKp26.2 with PEA128 and RSU-F9L while highlighting genomic distinctions consistent with its classification as a novel species. 3.3 Strain-dependent infection dynamics of SAKp26.2 revealed by spot assay lysis, EOP variability, and diverse killing patterns. Spot assays with phage SAKp26.2 demonstrated plaque formations at varying dilutions across different clinical isolates (Fig. S10) . The SAKp26.2 suspension produced plaques on the lawn of the U5877 (primary host) clinical strain of K. pneumoniae up to 10⁻⁷ dilution, with a surrounding halo around the core plaque, forming a characteristic bull’s-eye morphology. We skipped spotting the phage suspension from the 10 − 1 dilution because the titer was very high, and all the sensitive 16 strains showed a similar lysis pattern. Bull’s-eye plaques were observed up to 10⁻⁸ dilution on the lawn of O3933 indicating a strong lysis. On the lawn of U2150, plaques were visible up to 10⁻⁷ dilution of phage suspension, while the halo was observed up to 10⁻³ dilution. In O775, plaques were detected up to 10⁻⁵ dilution, with halo formation observed up to 10⁻² dilution. For P4272, plaques were observed up to a 10⁻⁸ dilution; however, the phage showed strong lysis up to a 10⁻³ dilution, whereas halo formation was visible up to 10⁻⁵ dilution. Isolate no U198 showed relatively poor phage susceptibility, with plaques detectable only up to 10⁻⁵ dilution and no halo formation. Similarly, in U7224, plaques were observed up to 10⁻⁵ dilution without any halo formation. In U6875, halo rings were visible up to 10⁻³ dilution, while plaques were observed up to 10⁻⁶ dilution. In U6795, halo rings were detected up to 10⁻³ dilution and plaques up to a 10⁻⁷ fold dilution. For U6350, a weak killing activity was observed up to 10⁻⁵ dilution. In B5521, halo rings were present up to 10⁻³ dilution, whereas distinct plaques were detected up to 10⁻⁶ dilution. In U5485, distinct halo rings were observed up to 10⁻³ dilution and plaques up to 10⁻⁷ dilution. Similarly, in P4725, halo rings were visible up to 10⁻³ dilution, while plaques were observed up to 10⁻⁶ dilution. In U4636, bull’s-eye plaques were observed up to 10⁻⁷ dilution. In U4444, halo rings were present up to10⁻³ dilution, with plaques detectable up to 10⁻⁶ dilution. Clinical isolate U4317 exhibited least susceptivity against phage SAKp26.2 and showed a few faint and indistinct clearing zones, indicating inefficient killing by the phage. To establish a reference host for phage SAKp26.2, the phage was amplified in three clinical isolates; O3933, P4725, and U5877. The amplified phage from three different strains were subsequently subjected to titer enumeration on each of the three clinical isolates to evaluate the lytic activity. When SAKp26.2 was amplified in the O3933 and used to infect the three isolates, plaques were observed up to 10⁻¹¹ dilution in O3933. Subsequently in strain P4725, plaques were detected up to 10⁻⁵ dilution, while in U5877 plaques were observed up to 10⁻¹⁰ dilution (Fig. S11) . Similarly, when the phage was amplified in the P4725 and tested on the same three hosts, plaques were detected up to 10⁻⁷, 10⁻¹⁰, and 10⁻⁶ dilutions in O3933, P4725, and U5877, respectively (Fig. S12) . In contrast, when SAKp26.2 was amplified in the U5877 clinical isolate and subjected to infect the three isolates, plaques were observed up to 10⁻¹¹, 10⁻¹⁰, and 10⁻¹⁰ dilutions in O3933, P4725, and U5877 indicating a similar degree of phage infection in all the strains (Fig. S13) . All experiments were performed with three biological and technical replicates. Comparative analysis of the efficiency of SAKp26.2 amplified in different hosts indicated that the phage amplified in the U5877 clinical isolate exhibited the most efficient plaque formation across the tested hosts. Therefore, the clinical isolate U5877 was selected as the reference host for phage SAKp26.2 The efficiency of plating (EOP) analysis ( Fig. 3 a ) revealed substantial variation in the replication efficiency of phage SAKp26.2 across different clinical isolates relative to the reference host U5877 [ 31 ]. Among the tested strains, O3933 exhibited the highest replication efficiency (EOP = 12.0), followed by U4636 (EOP = 3.5) and U6875 (EOP = 2.0), indicating strong phage propagation. Intermediate replication efficiency was observed in several isolates, including U5485 (EOP = 0.33), U4444 (EOP = 0.32), U6795 (EOP = 0.27), U4317 (EOP = 0.19), P4725 (EOP = 0.15), U2150 (EOP = 0.14), and O775 (EOP = 0.13). In contrast, U7224 exhibited low replication efficiency (EOP = 0.003), despite showing moderate susceptibility in the spot assay (Fig. S10) . Notably, U198 and U6350, which displayed moderate to weak plaque formation, showed no detectable phage replication (EOP = 0). Interestingly, P4272 also exhibited an EOP of 0 despite demonstrating relatively strong lytic activity in the spot assay. Overall, the EOP analysis highlights considerable heterogeneity in the replication efficiency of phage SAKp26.2 across clinical isolates, underscoring the importance of host-dependent variability in phage infectivity and propagation. The kill kinetics of phage SAKp26.2 were evaluated against 16 clinical isolates to assess the temporal dynamics of phage-mediated bacterial killing and the emergence of resistance. Monitoring changes in OD 600 over time enabled characterisation of host-specific responses, reflecting variations in phage adsorption, replication efficiency, and resistance development patterns. Resistance emergence pattern: clinical isolates U5877, O3933, P4272, U7224, and U4636 developed resistance at 240, 280, 120, 160, and 140 minutes post-infection, respectively ( Fig. 3 q, 3 p, 3 m, 3 k, 3 d ) . Following the emergence of resistance, the bacterial populations recovered and reached OD 600 levels comparable to the untreated bacterial control. Rise-decline-rise pattern: clinical isolates U2150, O775, U6795, B5521, and P4725 initially showed an increase in OD 600 , similar to the control. Upon initiation of phage killing, a sharp decline was observed, followed by another rise due to resistance emergence. In U2150, the initial increase in O.D. was observed at 40 minutes post-infection and continued until 180 minutes, after which a rapid decline was observed, followed by a rise after 320 minutes ( Fig. 3 o ) . Similarly, O775 showed an initial increase from 40 minutes. It persisted until 180 minutes, after which the OD 600 decreased to ~0.2 until 340 minutes. After 340 minutes, a second rise in O.D. was observed due to the emergence of a resistance subpopulation ( Fig. 3 n ) . U6795 demonstrated an initial rise at 40 minutes, followed by a decline at 180 minutes, and a second rise at 280 minutes post-infection ( Fig. 3 i ) . B5521 exhibited a similar pattern, with an initial rise at 40 minutes, a decline at 180 minutes, and a second rise at 280 minutes ( Fig. 3 g ) . In P4725, the first rise occurred at 40 minutes, followed by a decline at 180 minutes and a second rise at 320 minutes post-infection ( Fig. 3 e ) . None of these isolates reached the OD 600 levels of the bacterial control. Parallel-growth pattern: clinical isolates U6875, U5485, U4444, and U4317 treated with phage showed growth patterns in which the treated bacteria grew parallel to the untreated control, without any sharp decline. However, the O.D. of phage-treated bacteria never reached the O.D. of the control ( Fig. 3 j, 3 f, 3 c, 3 b ) . In all these isolates, resistance to the phage appeared within 40 minutes of infection. Weak infection: clinical isolates U198 and U6350 exhibited growth curves similar to those of the bacterial control, with only minor deviations in OD 600 following phage infection ( Fig. 3 l, 3 h ) . 3.4 SAKp26.2 suppresses bacterial growth at different MOIs and dismantles mature biofilm architecture The kill-kinetics assay indicated that the phage SAKp26.2 prevented bacterial growth for up to 5 hours at all tested MOIs ( Fig. 4 a ). SAKp26.2 showed efficient biofilm reduction post-phage treatment on 24-hour-old biofilm with ~ 99.0% and ~ 95.0% reduction in viable cell count within 4 hours and 8 hours of phage treatment, respectively ( Fig. 4 b ). In the biofilm biomass quantification by crystal violet staining assay, a significant reduction in OD595 was observed at 4 hours and 8 hours post-phage treatment. A ~ 2-fold decrease in optical density was seen after 8 hours of phage treatment ( Fig. 4 c ). Live-dead staining further confirmed the antibiofilm activity of phage SAKp26.2. Fluorescence imaging of untreated biofilms showed a dense population of viable bacterial cells predominantly stained green (SYTO9), indicating intact and metabolically active cells. In contrast, phage-treated biofilms exhibited a marked increase in red fluorescence (propidium iodide), representing membrane-compromised or dead bacterial cells ( Fig. 4 d- 4 e ). The treated biofilms displayed a significant reduction in green fluorescence along with dispersed clusters of red-stained cells, indicating extensive phage-mediated killing within the biofilm matrix. Three-dimensional surface reconstruction of the fluorescence images further demonstrated a substantial reduction in biofilm thickness and structural integrity following phage treatment. The untreated biofilm formed a dense and elevated architecture, whereas the phage-treated biofilm appeared flattened with reduced biomass and disrupted surface topology ( Fig. 4 f– 4 g ) . These observations corroborate the CFU reduction and crystal violet assay results, confirming the strong antibiofilm efficacy of phage SAKp26.2. 3.5 Phage-antibiotic treatment suppresses resistance emergence. In the case of ceftazidime treatment, bacterial resistance, indicated by an increase in OD 600 and visible turbidity, emerged approximately 19 h post-treatment at the highest antibiotic concentration (1.25 mg/mL) ( Fig. 5 a ). In contrast, treatment with phage SAKp26.2 alone delayed the emergence of bacterial resistance for up to 6 h across all tested MOIs (100–0.048) ( Fig. 5 b ). Notably, the phage–antibiotic combination demonstrated enhanced efficacy, where the highest antibiotic concentration (1.25 mg/mL) together with the highest phage MOI (100) prevented the development of bacterial resistance for the entire 24 h observation period ( Fig. 5 c ). The corresponding kill-kinetics curves are given in the supplementary material (Fig. S14, S15, S16). A more pronounced effect of the phage–antibiotic combination was observed with gentamicin. Bacterial resistance developed within 4 h of treatment across the tested antibiotic concentrations (1.25 mg/mL to 0.61 µg/mL) ( Fig. 5 d ) . In the combination treatment, sustained inhibition of bacterial resistance was observed for 24 h at three different antibiotic–phage combinations: 1.25 mg/mL with MOI 100, 0.625 mg/mL with MOI 50, and 0.3125 mg/mL with MOI 25 ( Fig. 5 e ). The corresponding kill-kinetics curves are given in the supplementary material (Fig. S17, S18) . Representative microtiter plate wells illustrating the antibiotic-phage combination assay are shown in the supplementary material (Fig. S19) , where wells with effective phage–antibiotic combinations (1.25 mg/mL with MOI 100, 0.625 mg/mL with MOI 50, and 0.3125 mg/mL with MOI 25) remain clear (OD 600 ≤ 0.225), indicating suppression of bacterial growth and resistance development. 3.6 Bacteriophage SAKp26.2 reduces biofilm of urinary tract catheter within 4 hours of treatment. The antibiofilm efficacy of phage SAKp26.2 was further evaluated against catheter-associated K. pneumoniae biofilms using a urinary tract catheter model ( Fig. 6 a ). Phage treatment resulted in a significant reduction in the number of viable biofilm-associated bacteria compared with the untreated control ( Fig. 6 b ). Approximately a ~ 99% decrease in bacterial survival was observed following 4 h of phage exposure, demonstrating the strong antibiofilm activity of SAKp26.2 against catheter-associated K. pneumoniae biofilms. Scanning electron microscopy (SEM) analysis of untreated catheter surfaces revealed a dense and well-established biofilm structure composed of numerous rod-shaped bacterial cells embedded within an extracellular polymeric matrix The bacterial cells were tightly adhered to the catheter surface, forming multilayered aggregates characteristic of mature biofilms ( Fig. 6 c ). In contrast, phage-treated catheter biofilms exhibited a marked disruption of the biofilm architecture after 4 h of treatment ( Fig. 6 d ). The treated surfaces showed a substantial reduction in bacterial cell density with scattered and distorted bacterial cells, indicating extensive phage-mediated lysis and detachment of the biofilm matrix. The structural integrity of the biofilm appeared severely compromised, with only a few residual bacterial cells remaining attached to the catheter surface. Collectively, these results indicate that phage SAKp26.2 effectively disrupts established catheter-associated biofilms by reducing bacterial viability and dismantling the structural integrity of the biofilm matrix. 4. Discussion The surge in the incidence of multidrug-resistant (MDR) Klebsiella pneumoniae infections, particularly those involving biofilm formation on medical devices, highlights the urgent need to develop novel therapeutic strategies [ 32 ]. In this study, a lytic bacteriophage, SAKp26.2, was isolated and characterised, which showed significant antibacterial and antibiofilm activity against clinical isolates of K. pneumoniae . The plaque morphology of SAKp26.2 showed distinct bullseye plaques with halos, indicating the presence of a depolymerase enzyme ( Fig. 1 a ) . The production of halos by phages is strongly associated with polysaccharide depolymerases, which degrade bacterial capsules and the biofilm matrix, allowing phages to infect biofilm-embedded bacteria [ 20 ]. Moreover, transmission electron microscopy confirmed that SAKp26.2 bears an icosahedral head with a short non-contractile tail ( Fig. 1 b ) , which is characteristic of phages belonging to the Autographiviridae family [ 29 ]. The observed high adsorption efficiency ( Fig. 1 c ) and short latent period ( Fig. 1 d ) are indicative of efficient host attachment and lytic replication efficiency of SAKp26.2 [ 33 ]. One of the major infections caused by K. pneumoniae is infection of the urinary tract [ 13 ], which has an average pH of 6.0 and temperature of 37°C [ 34 ]. For a phage to be effectively used as a therapeutic agent, it must be stable across a range of pH levels, temperatures, and solvents [ 35 ]. The phage remained stable over a pH range of 5–10 ( Fig. 1 e ) , at temperatures up to 55 ºC ( Fig. 1 f ) and in solvents such as 0.9% saline, PBS and chloroform ( Fig. 1 g ). Genomic analysis of SAKp26.2 showed that it has a compact linear dsDNA genome of size 41.4 kb (Fig. S4) with 50 coding sequences organised in functional modules typical of Autographviridae phages (supplementary table 2) . Importantly, no genes related to lysogeny, antibiotic resistance, or bacterial virulence were found in its genome, indicating its purely virulent nature and, thus, safe for therapeutic use (supplementary table 2 & Fig. 2 a ) . The presence of genes related to S-adenosyl-L-methionine hydrolase and serine/threonine kinases indicates strategies to bypass bacterial immune systems (supplementary table 2) , thereby enabling efficient infection of clinical isolates. Comparative genomics of SAKp26.2 revealed that it shares a very high level of similarity with Klebsiella phages RSU-F9L and PEA128 ( Fig. 2 e ) . The ANI value of the phage is below the 95% ICTV threshold; therefore, it is confirmed to be a new species of Autographiviridae (Fig. S8) . The infectivity and antibacterial potential of phage SAKp26.2 were assessed by host range determination, efficiency of plating (EOP), and kill kinetics analyses of clinical K. pneumoniae isolates. The killing dynamics and replication efficiency of the phage SAKp26.2 vary considerably across different clinical strains. The host range analysis showed that SAKp26.2 can infect multiple K. pneumoniae isolates, as observed by plaque formation across a wide range of phage dilutions. The phage SAKp26.2 produced plaques even at higher dilutions (10⁻⁷–10⁻⁸) on clinical isolates of K. pneumoniae , like U5877, O3933, U2150, U4636, U6795, and U5485, indicating strong phage infectivity and efficient propagation in these hosts. Reduced susceptibility and limited phage replication were observed in some isolates, such as U198, U7224, and U6350, along with plaque formation only at lower dilutions. The phage produced no plaque on the isolate U4317, with only faint clearing zones (Fig. S10) , implying inefficient phage-mediated killing in this isolate [ 27 ]. A characteristic bull’s-eye morphology of plaque was observed with a central core lysis zone surrounded by a halo during the host range analysis in several isolates (Fig. S10). Degradation of capsular polysaccharides of bacterial cells by phage-encoded depolymerases results in halo formation around plaques [ 24 ]. K. pneumoniae is known for its thick capsule, which can act as a barrier to phage adsorption [ 36 ]. Therefore, the presence of halos in isolates such as U5877, O3933, U6875, U6795, U5485, and P4725 suggests that SAKp26.2 may harbour enzymatic activity that degrades specific capsular structures, thus resulting in stronger infection [ 37 ]. Conversely, the absence of halos in isolates such as U198 and U7224 (Fig. S10) may indicate differences in capsular composition or reduced activity of the phage depolymerase against those capsule types. The efficiency of plating assay was conducted to quantify replication efficiency across different hosts and to further characterise phage infectivity. U5877 was chosen as the reference host considering the comparative amplification experiments, which showed that phage propagated in this isolate produced the most efficient plaque formation across multiple hosts (S11, S12, S13) . This observation highlights the effect of the propagation host on phage infectivity, as host-specific factors might affect phage particle maturation, adsorption efficiency, or receptor recognition [ 38 ][ 39 ]. The EOP analysis showed substantial variable phage replication efficiency among the tested isolates. High EOP values observed in O3933 and U4636 ( Fig. 3 a ) indicate that these isolates provide highly favourable conditions for phage replication, in some cases exceeding the efficiency observed in the reference host. Similarly, U6875 also supported efficient phage propagation. In contrast, several isolates including P4725, U5485, U6795, O775, U2150, U4444, and U4317 showed intermediate EOP values ( Fig. 3 a ) , indicating moderate susceptibility to the phage [ 31 ]. These intermediate levels of replication may reflect partial receptor compatibility or the presence of bacterial defense mechanisms that limit phage proliferation [ 40 ]. Extremely low EOP values in U7224 and the inefficient replication observed in U6350, U198, and P4272 ( Fig. 3 a ) further highlight the heterogeneity of phage-host interactions among clinical isolates. Kill kinetics experiments were performed to investigate the interaction dynamics between SAKp26.2 and its bacterial hosts. Post-phage infection, four distinct bacterial growth patterns were observed, reflecting differences in replication efficiency, susceptibility, and the emergence of resistance. In the classical lytic pattern, isolates such as U5877 ( Fig. 3 q ) , O3933 ( Fig. 3 p ) , P4272 ( Fig. 3 m ) , U7224 ( Fig. 3 k ) , and U4636 ( Fig. 3 d ) initially exhibited inhibition by the phage but eventually recovered as resistant populations emerged. The timing of resistance development differed among isolates, potentially due to the variations in mutation rates, receptor variability, or intracellular defence mechanisms. The incomplete recovery observed in O3933 ( Fig. 3 p ) indicates that the phage retained partial efficacy even after resistance developed. The isolates U2150 ( Fig. 3 o ) , O775 ( Fig. 3 n ) , U6795 ( Fig. 3 i ) , B5521 ( Fig. 3 g ) , and P4725 ( Fig. 3 e ) showed a rise–dip–rise growth pattern, implying complex associations between bacterial growth and phage replication. The bacterial populations initially increased, then declined due to active phage-mediated lysis, and regrew as resistant or partially resistant cells emerged. Interestingly, these isolates did not reach the optical density of the untreated control, indicating that phage SAKp26.2 continued to exert inhibitory effects on bacterial growth even after regrowth. Isolates such as U6875 ( Fig. 3 j ) , U5485 ( Fig. 3 f ) , U4444 ( Fig. 3 c ) , and U4317 ( Fig. 3 b ) showed a parallel-rise pattern, in which bacterial growth increased along with the control but remained consistently lower than the control. Resistance emerged rapidly within 40 minutes, suggesting either intrinsic resistance mechanisms or rapid adaptation to phage exposure. Meanwhile, isolates U198 ( Fig. 3 l ) and U6350 ( Fig. 3 h ) showed only minor deviations from the control growth curve, indicating minimal susceptibility to SAKp26.2 infection, consistent with their low plaque formation and inefficient EOP values. The combined analyses of host range, efficiency of plating (EOP), and kill kinetics underscore the complex and strain-specific interactions between SAKp26.2 and clinical K. pneumoniae isolates. Although SAKp26.2 can infect multiple strains and replicate efficiently in certain hosts, differences in infection efficiency and the rapid emergence of resistant populations exhibit significant challenges for single-phage therapeutic applications. These results indicate that, given SAKp26.2's considerable antibacterial potential, its most effective clinical use may involve incorporating it into a phage cocktail or phage-antibiotic combination therapy to expand host coverage and delay resistance emergence. In the present study, resistance to ceftazidime emerged approximately 19 hours after treatment (at the highest concentration) and around 3 hours post-treatment at other concentrations ( Fig. 5 a ) , while resistance to gentamicin developed within 4 hours across the tested concentrations ( Fig. 5 d ) . These findings demonstrate the robust adaptive capacity of K. pneumoniae under antibiotic selective pressure, which is enabled by multiple resistance mechanisms and the rapid acquisition of adaptive mutations [ 41 , 42 ]. Administration of phage SAKp26.2 alone delayed the development of bacterial resistance for up to 6 hours ( Fig. 5 b ) , suggesting sustained lytic activity against the host strain. However, the most pronounced antibacterial effect was observed when the phage was combined with antibiotics, and several phage-antibiotic combinations suppressed bacterial regrowth and prevented the development of resistance throughout the 24-hour observation period ( Fig. 5 c, 5 e ) . Phage-antibiotic combinations are increasingly recognised for their capacity to delay or suppress the emergence of resistance, which constitutes a significant advantage of combination therapy. In these systems, antibiotics reduce bacterial growth and impose selective pressure, while bacteriophages infect and lyse susceptible bacterial cells, thereby restricting the expansion of resistant subpopulations [ 43 ][ 44 ]. The enhanced suppression of resistance observed in combination treatments arises from the complementary mechanisms of phages and antibiotics. Antibiotics inhibit essential cellular processes, whereas bacteriophages replicate within bacterial hosts and induce cell lysis, continuously reducing bacterial population density. This dual selective pressure reduces the probability of the resistant mutants emerging in the population. Furthermore, antibiotic-induced physiological stress can enhance bacterial susceptibility to phage infection, thereby contributing to the suppression of resistant populations [ 45 ]. Notably, gentamicin–phage combinations exhibited a more substantial inhibition of resistance (Fig. S18) compared to ceftazidime (Fig. S16). Aminoglycosides such as gentamicin disrupt protein synthesis by targeting the 30S ribosomal subunit, resulting in bactericidal activity and cellular stress that may enhance phage-mediated killing. Similar effects of phage-antibiotic combinations have also been reported against multidrug-resistant Gram-negative pathogens, including ESBL-producing and carbapenem-resistant Escherichia coli [ 46 ]. Furthermore, the observed suppression of bacterial regrowth at higher phage multiplicities of infection (MOIs) suggests that increasing phage pressure can further limit the expansion of resistant subpopulations. Higher MOIs increase the likelihood of phage-host encounters, thereby accelerating bacterial killing and reducing the probability that resistant mutants will proliferate. Previous studies have also demonstrated that the extent of phage-antibiotic synergy depends on the stoichiometric relationship between phage particles, bacterial cells, and antibiotic concentration, highlighting the importance of optimizing treatment parameters when evaluating phage-antibiotic combination therapy [ 47 ]. A major finding of this study is the strong antibiofilm activity of SAKp26.2. Biofilm-associated infections represent a major clinical challenge because biofilms provide bacteria with protection against antibiotics and host immune responses. Treatment of established biofilms with SAKp26.2 resulted in substantial reductions in viable cell counts and biofilm biomass ( Fig. 4 b, 4 c ) . Fluorescent live–dead imaging ( Fig. 4 d, 4 e ) further confirmed extensive phage-mediated killing within the biofilm matrix and significant disruption of biofilm architecture. Three-dimensional reconstruction of the biofilm structures revealed marked reductions in thickness and structural complexity following phage treatment ( Fig. 4 f, 4 g ) . These observations are consistent with previous reports showing that phages encoding depolymerases can penetrate and degrade biofilm matrices, thereby enhancing bacterial killing [ 48 ]. The ability of SAKp26.2 to disrupt biofilms was further validated in a catheter-associated biofilm model. SEM analysis demonstrated that untreated catheter surfaces were heavily colonized by multilayered bacterial communities embedded within extracellular polymeric substances ( Fig. 6 c ) . In contrast, phage treatment resulted in substantial disruption of the biofilm structure and a dramatic reduction in viable bacterial cells within only four hours ( Fig. 6 b, 6 d ) . Since catheter-associated infections represent a major source of hospital-acquired infections [ 49 ], these findings highlight the potential of SAKp26.2 as a candidate for controlling device-associated K. pneumoniae biofilms [ 50 ]. 5. Conclusion In this study, bacteriophage SAKp26.2 was isolated and characterized as a novel lytic phage infecting clinical K. pneumoniae strains. SAKp26.2 belongs to the family Autographviridae , which is confirmed by morphological and genomic analyses, and it has a compact modular genome of 41.4 kb. Since the SAKp26.2 genome lacks the genes associated with lysogeny, antibiotic resistance, or virulence, it is suitable for therapeutic applications. Comparative genomic and phylogenetic analysis further supported that SAKp26.2 is a novel species of the Autographviridae lineage. SAKp26.2 can infect multiple clinical K. pneumoniae , however, differences between spot assay lysis, EOP value, and kill-kinetics indicate that clear zone formation alone does not necessarily reflect productive phage infection. These findings suggest that to precisely evaluate phage infectivity and replication efficiency, a combination of spot assays and quantitative approaches, such as EOP and kill kinetics, is important. Also, SAKp26.2 exhibited strong antibacterial and antibiofilm activity against K. pneumoniae. The phage was effective in suppressing bacterial growth, significantly reducing biofilm biomass, and disrupting the architecture of mature biofilms in both microtiter plate and catheter-associated biofilm models. Furthermore, treatments with phage-antibiotic combination enhanced bactericidal activity and delayed the emergence of resistance. Thus, these findings demonstrate that bacteriophage SAKp26.2 possesses several characteristics desirable for a therapeutic phage, including a rapid infection, strong antibiofilm activity, genomic safety, and synergistic potential with antibiotics. These properties of SAKp26.2 make it a promising candidate for the control of MDR K. pneumoniae infections, particularly those associated with biofilms and medical devices. Declarations Acknowledgements : A. and SC would like to thank DBT for the fellowship support. AC would like to thank ICMR for the funding support. Author Contributions: AC and SC contributed to the study conception and design. Material preparation, data collection and analysis were performed by A., DCS, and AJ. The first draft of the manuscript was written by A., SC and DCS. The manuscript was reviewed by AC and RK. AC contributed supervision to resources, project management and grant acquisition. All authors read and approved the final manuscript. Funding We gratefully acknowledge the financial support provided by ICMR Data availability The datasets generated and/or analysed during the current study are available at NCBI Gene Bank with accession No PX974311. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests. References World Health Organization. (2001). WHO global strategy for containment of antimicrobial resistance (No. WHO/CDS/CSR/DRS/2001.2). World Health Organization. Ashurst JV, Dawson A. Klebsiella Pneumonia. [Updated 2023 Jul 20]. In: StatPearls [Internet]. 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K., Pickard, D. J., Wilksch, J. J., Schittenhelm, R. B., Strugnell, R. A., Dougan, G., & Lithgow, T. (2021). Mechanistic Insights into the Capsule-Targeting Depolymerase from a Klebsiella pneumoniae Bacteriophage. Microbiology spectrum , 9 (1), e0102321. https://doi.org/10.1128/Spectrum.01023-21 Fischetti V. A. (2008). Bacteriophage lysins as effective antibacterials. Current opinion in microbiology , 11 (5), 393–400. https://doi.org/10.1016/j.mib.2008.09.012. Cui, L., Watanabe, S., Miyanaga, K., Kiga, K., Sasahara, T., Aiba, Y., Tan, X. E., Veeranarayanan, S., Thitiananpakorn, K., Nguyen, H. M., & Wannigama, D. L. (2024). A Comprehensive Review on Phage Therapy and Phage-Based Drug Development. Antibiotics (Basel, Switzerland) , 13 (9), 870. https://doi.org/10.3390/antibiotics13090870. Ibrahim, R., & Aranjani, J. M. (2026). Bacterial defense mechanisms against bacteriophages: an evolutionary arms race. 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Frontiers in pharmacology , 15 , 1356179. https://doi.org/10.3389/fphar.2024.1356179. Tagliaferri, T. L., Jansen, M., & Horz, H. P. (2019). Fighting Pathogenic Bacteria on Two Fronts: Phages and Antibiotics as Combined Strategy. Frontiers in cellular and infection microbiology , 9 , 22. https://doi.org/10.3389/fcimb.2019.00022. Shamsuzzaman, M., Kim, S., & Kim, J. (2025). Therapeutic potential of novel phages with antibiotic combinations against ESBL-producing and carbapenem-resistant Escherichia Coli. Journal of global antimicrobial resistance , 43 , 86–97. https://doi.org/10.1016/j.jgar.2025.04.005. Gu Liu, C., Green, S. I., Min, L., Clark, J. R., Salazar, K. C., Terwilliger, A. L., Kaplan, H. B., Trautner, B. W., Ramig, R. F., & Maresso, A. W. (2020). Phage-Antibiotic Synergy Is Driven by a Unique Combination of Antibacterial Mechanism of Action and Stoichiometry. mBio , 11 (4), e01462-20. https://doi.org/10.1128/mBio.01462-20. Guo, Z., Liu, M., & Zhang, D. (2023). Potential of phage depolymerase for the treatment of bacterial biofilms. Virulence , 14 (1), 2273567. https://doi.org/10.1080/21505594.2023.2273567. Rubi, H., Mudey, G., & Kunjalwar, R. (2022). Catheter-Associated Urinary Tract Infection (CAUTI). Cureus , 14 (10), e30385. https://doi.org/10.7759/cureus.30385. Curtin, J. J., & Donlan, R. M. (2006). Using bacteriophages to reduce formation of catheter-associated biofilms by Staphylococcus epidermidis. Antimicrobial agents and chemotherapy , 50 (4), 1268–1275. https://doi.org/10.1128/AAC.50.4.1268-1275.2006. Additional Declarations No competing interests reported. Supplementary Files SAKp26.2AafreenetalArticleSupplementaryFile.docx Graphicalabstract.jpg Graphical abstract Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 08 May, 2026 Reviews received at journal 04 May, 2026 Reviews received at journal 01 May, 2026 Reviews received at journal 28 Apr, 2026 Reviews received at journal 26 Apr, 2026 Reviewers agreed at journal 22 Apr, 2026 Reviewers agreed at journal 22 Apr, 2026 Reviewers agreed at journal 22 Apr, 2026 Reviewers agreed at journal 18 Apr, 2026 Reviewers agreed at journal 18 Apr, 2026 Reviews received at journal 18 Apr, 2026 Reviewers agreed at journal 17 Apr, 2026 Reviewers agreed at journal 17 Apr, 2026 Reviewers agreed at journal 17 Apr, 2026 Reviewers agreed at journal 12 Apr, 2026 Reviewers invited by journal 06 Apr, 2026 Editor assigned by journal 25 Mar, 2026 Submission checks completed at journal 20 Mar, 2026 First submitted to journal 20 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9111241","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":621624987,"identity":"6f44b28e-1877-4a76-bdcd-0f93575f62c3","order_by":0,"name":"Aafreen .","email":"","orcid":"","institution":"University of Delhi","correspondingAuthor":false,"prefix":"","firstName":"Aafreen","middleName":"","lastName":".","suffix":""},{"id":621624990,"identity":"d7518f0c-6377-47df-8e09-2d7ff25edb01","order_by":1,"name":"Sambuddha Chakraborty","email":"","orcid":"","institution":"University of Delhi","correspondingAuthor":false,"prefix":"","firstName":"Sambuddha","middleName":"","lastName":"Chakraborty","suffix":""},{"id":621624991,"identity":"627246bf-981c-4f86-afcd-5965f34c312a","order_by":2,"name":"Deepangkar Chisim Sangma","email":"","orcid":"","institution":"University of Delhi","correspondingAuthor":false,"prefix":"","firstName":"Deepangkar","middleName":"Chisim","lastName":"Sangma","suffix":""},{"id":621624994,"identity":"e2954463-86fa-4f3e-a251-646792e8f31b","order_by":3,"name":"Anusha Jatley","email":"","orcid":"","institution":"University of Delhi","correspondingAuthor":false,"prefix":"","firstName":"Anusha","middleName":"","lastName":"Jatley","suffix":""},{"id":621624996,"identity":"c69aaf2d-f1af-425c-a75c-8f2e779edd89","order_by":4,"name":"Ram Karan","email":"","orcid":"","institution":"University of Delhi","correspondingAuthor":false,"prefix":"","firstName":"Ram","middleName":"","lastName":"Karan","suffix":""},{"id":621624998,"identity":"936b963c-a7f1-4400-b735-ce4cd37e4710","order_by":5,"name":"Ashwini Chauhan","email":"data:image/png;base64,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","orcid":"","institution":"University of Delhi","correspondingAuthor":true,"prefix":"","firstName":"Ashwini","middleName":"","lastName":"Chauhan","suffix":""}],"badges":[],"createdAt":"2026-03-13 06:55:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9111241/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9111241/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106773738,"identity":"c02ef32e-c40d-4e4b-a1e5-0fcd958edcc5","added_by":"auto","created_at":"2026-04-13 10:36:50","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":124645,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComprehensive Biological Characterization of the Bacteriophage SAKp26.2 (a) \u003c/strong\u003ePlaque morphology of SAKp26.2 on the lawn of host \u003cem\u003eK. pneumoniae \u003c/em\u003eU5877 showing depolymerase activity around the core lysis zone (0.20 mm) \u003cstrong\u003e(b) \u003c/strong\u003eTEM image of phage with an icosahedral head (diameter ~50 nm), and a short tail. \u003cstrong\u003e(c) \u003c/strong\u003eAdsorption assay of phage with host, indicating 95% adsorption within 5 minutes. \u003cstrong\u003e(d) \u003c/strong\u003eOne-step growth curve of phage exhibiting a latency period of 10 minutes and a burst size of around 166 for each host cell. \u003cstrong\u003e(e) \u003c/strong\u003epH stability of SAKp26.2 – the phage remained relatively stable from pH 5 to 9, while complete degradation was seen at pH 1, 3, and 14. \u003cstrong\u003e(f) \u003c/strong\u003eTemperature stability of SAKp26.2 – the phage remained stable at temperatures from 37 ºC to 55 ºC, while completely degraded at 85 ºC to 100 ºC. \u003cstrong\u003e(g) \u003c/strong\u003eSolvent stability of SAKp26.2 – the phage retained stability in 0.9% saline, PBS, and chloroform. GraphPad Prism v8.0.1 was used to produce the graphs.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9111241/v1/068661add312776bc27ab96f.jpg"},{"id":106960510,"identity":"9a56b1a1-afcb-47b8-a0c2-a601149ff965","added_by":"auto","created_at":"2026-04-15 09:21:33","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":186753,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenomic organization and phylogenetic analysis of phage SAKp26.2.\u003c/strong\u003e \u003cstrong\u003e(a)\u003c/strong\u003e Circular genome map showing the distribution and orientation of predicted coding sequences across the 41,467 bp genome of SAKp26.2. \u003cstrong\u003e(b)\u003c/strong\u003eWhole-proteome phylogenetic tree (VipTree) indicating the placement of SAKp26.2 within the \u003cem\u003eAutographiviridae\u003c/em\u003e lineage infecting members of Pseudomonadota. \u003cstrong\u003e(c)\u003c/strong\u003e Terminase large subunit–based phylogenetic tree showing the evolutionary relationship of SAKp26.2 with closely related \u003cem\u003eKlebsiella\u003c/em\u003ephages. \u003cstrong\u003e(d)\u003c/strong\u003e Intergenomic similarity heatmap depicting pairwise nucleotide identity among SAKp26.2 and related phages. \u003cstrong\u003e(e)\u003c/strong\u003e Comparative genome alignment of SAKp26.2 with \u003cem\u003eKlebsiella\u003c/em\u003e phage PEA128 and Klebsiella phage RSU-F9L showing conserved genomic synteny with regions of divergence.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9111241/v1/df325e80a7878a174764e798.jpg"},{"id":106773742,"identity":"ebb3d400-3e56-462a-b643-e43a401ff879","added_by":"auto","created_at":"2026-04-13 10:36:50","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":548108,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEfficiency of plating (EOP), and killing kinetics of phage SAKp26.2 against clinical \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eK. pneumoniae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e isolates\u003c/strong\u003e \u003cstrong\u003e(a) \u003c/strong\u003eEOP of SAKp26.2 on clinical isolates O3933, P4272, U4317, U4444, U4636, P4725, U5485, B5521, U6350, U6795, U6875, U7224, U198, O775, and U2150, using U5877 as the reference host. High replication efficiency was observed in O3933 (EOP = 12.0), U4636 (EOP = 3.5), and U6875 (EOP = 2.0). Intermediate replication efficiency was detected in P4725 (0.15), U5485 (0.33), U6795 (0.27), O775 (0.13), and U2150 (0.14). Low replication efficiency was observed in U7224 (0.003), whereas inefficient replication was detected in U6350, U198, and P4272. \u003cstrong\u003e(b–q)\u003c/strong\u003e Kill-kinetics analysis of SAKp26.2 on different clinical isolates based on OD\u003csub\u003e600\u003c/sub\u003e measurements. U4317, U4444, U5485, and U6875 exhibited OD\u003csub\u003e600\u003c/sub\u003e increases parallel to the control with resistance emerging before 40 min of infection. U4636 developed resistance at 140 min without reaching control OD\u003csub\u003e600 \u003c/sub\u003elevels. U7224 and P4272 acquired resistance at 160 min and 120 min, respectively, with OD\u003csub\u003e600\u003c/sub\u003e eventually reaching control levels. O3933 and U5877 developed resistance at 280 min and 240 min, respectively, after which OD600 rapidly reached the control. P4725, B5521, U6795, O775, and U2150 showed a characteristic rise–dip–rise pattern in OD\u003csub\u003e600\u003c/sub\u003e, with reduced growth compared with the control after the second rise. U6350 and U198 displayed OD\u003csub\u003e600\u003c/sub\u003e increases similar to the control with only minor deviations. GraphPad Prism v8.0.1 was used to produce the graphs.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9111241/v1/b02ce5ffdea3820b554ae557.jpg"},{"id":106960784,"identity":"287bfa31-0d04-44f9-bc89-747898e14c49","added_by":"auto","created_at":"2026-04-15 09:23:07","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":85354,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntibacterial and antibiofilm activity of phage SAKp26.2 against MDR\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e K. pneumoniae\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e \u003cstrong\u003e(a)\u003c/strong\u003e Killing kinetics of phage SAKp26.2 at different multiplicities of infection (MOI), showing suppression of bacterial growth compared with the untreated control. \u003cstrong\u003e(b)\u003c/strong\u003e Reduction in viable biofilm-associated bacterial cells following phage treatment of 24 h preformed biofilms, expressed as percent survival relative to untreated control after 4 h and 8 h treatment. \u003cstrong\u003e(c)\u003c/strong\u003e Quantification of biofilm biomass by crystal violet staining showing significant reduction in optical density (OD595) following phage treatment. \u003cstrong\u003e(d–e)\u003c/strong\u003e Live/dead fluorescence microscopy of biofilms where green fluorescence represents viable bacterial cells (SYTO9) and red fluorescence represents membrane-compromised or dead cells (propidium iodide), demonstrating increased cell death after phage treatment. \u003cstrong\u003e(f–g)\u003c/strong\u003eThree-dimensional surface reconstruction of biofilm architecture illustrating dense biofilm structure in the control and reduced biofilm thickness and biomass following phage treatment. Data represented are means ± standard error of the mean of at least 3 independent experiments. *P \u0026lt; 0.05, **P \u0026lt; 0.005, ***P \u0026lt; 0.0005, ****P \u0026lt; 0.0001. GraphPad Prism v8.0.1 was used to produce the graphs.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9111241/v1/c5a988bbc87474b3b6279b51.jpg"},{"id":106773744,"identity":"71b2e70b-844e-422e-817c-b055c9c62fec","added_by":"auto","created_at":"2026-04-13 10:36:50","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":164433,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhage Antibiotic Treatment Suppresses Resistance Emergence. (a) \u003c/strong\u003eCeftazidime treatment alone showed emergence of bacterial resistance after ~19 h at the highest concentration (1.25mg/mL).\u003cstrong\u003e (b) \u003c/strong\u003ePhage SAKp26.2 treatment alone inhibited bacterial growth for approximately 6 h across all tested MOIs (100 to 0.048).\u003cstrong\u003e (c) \u003c/strong\u003eCombined treatment with ceftazidime and phage SAKp26.2 prevented the emergence of resistance for up to 24 h at the highest ceftazidime concentration and MOI. \u003cstrong\u003e(d)\u003c/strong\u003e Gentamicin treatment alone showed rapid emergence of resistance within ~4 h.\u003cstrong\u003e (e) \u003c/strong\u003eGentamicin-phage combination demonstrated prolonged suppression of bacterial growth for up to 24 h at specific antibiotic-phage combinations (1.25 mg/mL with MOI 100, 0.625 mg/mL with MOI 50, and 0.3125 mg/mL with MOI 25). GraphPad Prism v8.0.1 was used to produce the graphs.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9111241/v1/5a77d8935795a41ea0a8eafc.jpg"},{"id":106960789,"identity":"05361ee0-2401-4d0f-a48e-130b0b6c6dd9","added_by":"auto","created_at":"2026-04-15 09:23:08","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":131864,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntibiofilm activity of phage SAKp26.2 against catheter-associated \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eK. pneumoniae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e biofilms. \u003c/strong\u003eScanning electron microscopy (SEM) images showing the architecture of biofilms formed on urinary tract catheter surfaces. \u003cstrong\u003e(a)\u003c/strong\u003e Untreated control exhibiting dense bacterial colonization and well-developed biofilm matrix on the catheter surface. \u003cstrong\u003e(b)\u003c/strong\u003ePhage-treated biofilm after 4 h of exposure to SAKp26.2 showing disrupted biofilm structure, reduced bacterial density, and evidence of bacterial cell lysis. \u003cstrong\u003e(c) \u003c/strong\u003eQuantification of viable biofilm-associated bacterial cells demonstrating significant reduction in bacterial survival following phage treatment compared to the untreated control. \u003cstrong\u003eScale bar: 1 µm. \u003c/strong\u003eData represented are means ± standard error of the mean of at least 3 independent experiments. *P \u0026lt; 0.05, **P \u0026lt; 0.005, ***P \u0026lt; 0.0005, ****P \u0026lt; 0.0001. GraphPad Prism v8.0.1 was used to produce the graphs.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9111241/v1/d7a1f48a7ab12da76a22eb57.jpg"},{"id":106963007,"identity":"b52c2ca1-073c-467d-9174-49230740d847","added_by":"auto","created_at":"2026-04-15 09:41:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3208206,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9111241/v1/81d17aee-c082-4131-bf07-11772bb4fb0e.pdf"},{"id":106960076,"identity":"a7887cfc-cdf4-436e-8a59-cbf30f360774","added_by":"auto","created_at":"2026-04-15 09:18:35","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7515350,"visible":true,"origin":"","legend":"","description":"","filename":"SAKp26.2AafreenetalArticleSupplementaryFile.docx","url":"https://assets-eu.researchsquare.com/files/rs-9111241/v1/c91841700e152df8311edd5a.docx"},{"id":106773740,"identity":"1a25db47-640c-4549-862e-e57084a15869","added_by":"auto","created_at":"2026-04-13 10:36:50","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":149681,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract\u003c/p\u003e","description":"","filename":"Graphicalabstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9111241/v1/7a3539dcafa1fa105143375b.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genomic and translational characterisation of Autographviridae phage SAKp26.2 for catheter-associated biofilm clearance in drug-resistant Klebsiella pneumoniae","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAntimicrobial resistance (AMR) has emerged in the current century as one of the major contributors to public health challenges. The rapid spread of resistance renders conventional antibiotic treatment ineffective for adult healthy individuals but severely limits its effectiveness for most hospital-acquired bacterial infections in immunocompromised patients. Realising the urgency, the World Health Organization (WHO) has highlighted the necessity for an improvement and coordinated global effort to contain AMR especially for ESKAPE pathogens [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e is one of the ESKAPE group pathogens that acquires antibiotic resistance at an alarming rate. In 1929, resistance to beta-lactam antibiotics in gram-negative was discovered by Alexander Fleming. Since then, extensive studies have been done about \u003cem\u003eK. pneumoniae\u003c/em\u003e and was found that it produces a beta-lactamase that causes hydrolysis of beta-lactam ring in antibiotics hence resulting in an intrinsic resistance to cell wall synthesis targeting antibiotics like ampicillin and first-generation penicillin [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In 1983 and 1989 cases of Extended-spectrum beta-lactamase (ESBL) \u003cem\u003eK. pneumoniae\u003c/em\u003e were reported. ESBLs can hydrolyse oxyimino cephalosporins significantly reducing the efficiency of treatments with third-generation cephalosporins. This led to carbapenems as an effective antibiotic against ESBLs [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In course of time negligent application and disposal of antibiotic made bacteria adapt to carbapenems as well. \u003cem\u003eK. pneumoniae\u003c/em\u003e uses human as their primary reservoir, mainly colonizing in patient\u0026rsquo;s gastrointestinal tract and the hands of hospital personals. Higher numbers of such cases have been reported in South Asia [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e][\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and more importantly the mortality rate of patients with pneumonia caused by \u003cem\u003eK. pneumoniae\u003c/em\u003e is about 50% [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eK. pneumoniae\u003c/em\u003e passes on to their next generation the antimicrobial resistant genes through mobile elements such as plasmids, transposons and integrons. These elements play critical role in vertical and horizontal transmission of the genes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These might confer advantage against antibiotic by producing specified enzymes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], decreased cell permeability by cutting-off Omps [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], overexpression of efflux pumps such as KpnGH [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and modifying the key target of the antimicrobial agent [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Along with these features some \u003cem\u003eK. pneumoniae\u003c/em\u003e is capable of biofilm formation further exacerbating its clinical impact [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Infections associated with biofilms are extremely difficult to deal with. The resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e biofilms manage to survive in healthcare environments and on the devices used for treatment enhancing their infectivity [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. One such matter is the formation of biofilm by \u003cem\u003eK. pneumoniae\u003c/em\u003e on the catheter of patients with urinary tract infection (UTI). Each day a catheter remains indwelled in a patient, the risk of biofilm formation on the wall of catheter increases 3.55 times, and with 3 days the chances of UTI become 90% [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The effective way to counter such is to daily evaluation of patient for early catheter removal but many patients with UTIs associated with catheters cannot have their removed due to their underlying disease conditions [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. With such limitations on patients along with rapid increase in the acquired resistance and failure of conventional antibiotics, the development of novel antimicrobial strategies is what humanity requires.\u003c/p\u003e \u003cp\u003eTo limit the impact of AMR is of utmost importance, as the present figure of cases of antimicrobial-resistant-bacterial infections indicates arrival of a pandemic [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Novel countermeasures are required to be developed against AMR since, it is an unavoidable phenomenon, which renders the drug\u0026rsquo;s effectiveness [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. An alternate to antibiotics might keep the AMR in-check, as inappropriate usage and exposure of antibiotic is resulted in huge disease burden and accelerated emergence of AMR, and has shorten the list of effective antibiotics.\u003c/p\u003e \u003cp\u003eIn face of this challenge, bacteriophages have re-emerged as promising therapeutic candidates. Phage therapy, which predates the antibiotic era, offers several advantages, including high specificity toward bacterial hosts [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], self-amplification at the site of infection [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and the ability to target antibiotic-resistant bacteria [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Importantly, certain bacteriophages encode depolymerases and lytic enzymes capable of degrading bacterial capsules and biofilm matrices [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], making them particularly preferrable for treating biofilm-associated infections. Furthermore, combining bacteriophages with antibiotics has shown potential to enhance antibacterial efficacy [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and delay the emergence of resistance, suggesting a synergistic therapeutic approach [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we report the isolation and characterization of SAKp26.2, a novel lytic bacteriophage belonging to the family \u003cem\u003eAutographviridae\u003c/em\u003e, isolated from hospital sewage. We evaluated its antibacterial and antibiofilm activity against a panel of clinically relevant, drug-resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e strains. In addition to genomic characterization, we investigated its biofilm clearance capacity, and efficiency, and highlighted the correlation between efficiency of plating and kill kinetics which shows a very interesting result that can be mirrored in clinical setups. With clinically relevant infection catheter-associated biofilms, this work aims to advance the translational potential of phage-based therapeutics and contribute to the development of effective strategies against multidrug-resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Bacterial Strains and Growth Conditions\u003c/h2\u003e \u003cp\u003eThe multi drug-resistant (MDR) \u003cem\u003eK. pneumoniae\u003c/em\u003e strains were acquired from Madras Medical Mission Hospital (MMMH) in Chennai, India, were cultured at 37 \u0026ordm;C in Luria-Bertani Broth with shaking at 150 rpm, unless otherwise stated. Viable cell counts were estimated by spotting serial dilutions of the culture onto LB agar plates and incubating at 37\u0026deg;C overnight. The number of colony-forming units per mL was determined. All experiments were conducted in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Isolation and Purification of Bacteriophage\u003c/h2\u003e \u003cp\u003eThe bacteriophage SAKp26.2 was isolated from sewage water collected at the Madras Medical Mission Hospital (MMMH), Chennai, India, following the protocol described by Chakraborty \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Briefly, 5 mL of mid-log-phase XDR \u003cem\u003eK. pneumoniae\u003c/em\u003e clinical isolate U5877, grown to an OD\u003csub\u003e600\u003c/sub\u003e of 0.3\u0026ndash;0.6, was added to 5 mL of sterile LB medium. The mixture was incubated overnight at 37\u0026deg;C with shaking at 150 rpm, then centrifuged at 12,000\u0026times; g for 10 min at room temperature. The presence of lytic bacteriophages in the supernatant was preliminarily confirmed by a spot test, in which 10 \u0026micro;L of the supernatant was spotted onto the top of the lawn of the host bacterial isolate and allowed to dry. The plate was incubated at 37 \u0026ordm;C overnight. A clear zone of lysis suggests positive bacteriolytic activity.\u003c/p\u003e \u003cp\u003eA single phage was purified using a double agar overlay technique. The supernatant was serially diluted tenfold. 3 mL of 0.5% soft agar containing 100 \u0026micro;L of diluted supernatant and 500 \u0026micro;L of an overnight culture of \u003cem\u003eK. pneumoniae\u003c/em\u003e was poured onto a 1.5% LB agar plate and incubated overnight at 37\u0026deg;C. The next day, a single plaque was picked, inoculated into an early-log-phase culture, and incubated overnight. The suspension was centrifuged at 12,000\u0026times; g for 10 min at room temperature, and the supernatant was used for a double-layer agar overlay experiment. This procedure was repeated three times to get a single purified phage. Finally, the supernatant was treated with chloroform in a 1:10 ratio for 15 minutes. After chloroform treatment, the suspension was centrifuged, filtered through a 0.22 \u0026micro;m syringe-driven filter, and stored at 4\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Whole genome sequencing and bioinformatic analysis of SAKp26.2\u003c/h2\u003e \u003cp\u003eThe phage suspension was treated with DNase (1 \u0026micro;g/mL) and RNase (1 \u0026micro;g/mL) for 1 h to eliminate any extracellular bacterial DNA and RNA. The mixture was subsequently heated at 80\u0026deg;C for 15 min to inactivate the enzymes. After enzyme inactivation, 50 \u0026micro;g/mL proteinase K and 5% SDS were added to the phage suspension and incubated for 1 h at 56\u0026deg;C to denature the phage capsid proteins. The suspension was then mixed with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) and centrifuged at 13,000 \u0026times; g for 5 min at 4\u0026deg;C. The aqueous supernatant was carefully collected and mixed with an equal volume of chilled isopropanol, followed by incubation at \u0026minus;\u0026thinsp;20\u0026deg;C for 1 h to precipitate the phage DNA. The mixture was centrifuged to pellet the DNA, and the resulting pellet was washed with 1 mL of 75% ethanol and centrifuged at 12,000 \u0026times; g for 10 min at 4\u0026deg;C. The DNA pellet was then air-dried, resuspended in 30 \u0026micro;L of ddH₂O, and stored at \u0026minus;\u0026thinsp;20\u0026deg;C until further use. The concentration and purity of the extracted DNA were assessed using a Qubit 4.0 Fluorometer, and DNA integrity was verified on a 0.8% agarose gel prepared in 1\u0026times; TAE buffer.\u003c/p\u003e \u003cp\u003eWhole-genome sequencing of phage SAKp26.2 was performed using the Illumina HiSeq platform. Raw sequencing reads were subjected to quality control using Fastp (version 0.20.1), a multithreaded C\u0026thinsp;+\u0026thinsp;+\u0026thinsp;based tool. De novo genome assembly was performed using the St. Petersburg genome assembler (SPAdes) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bio.tools/spades\u003c/span\u003e\u003cspan address=\"https://bio.tools/spades\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The phage sequence was extracted using PHASTER. Genome annotation was initially carried out using Prokka (version 1.14.6) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://proksee.ca/tools/prokka\u003c/span\u003e\u003cspan address=\"https://proksee.ca/tools/prokka\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), followed by further functional annotation using additional tools including the RAST server version 2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://RAST.nmpdr.org\u003c/span\u003e\u003cspan address=\"http://RAST.nmpdr.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), BLASTp (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), InterPro (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/interpro/\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/interpro/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), UniProt (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and GenomeVx (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://conantlab.org/GenomeVx/\u003c/span\u003e\u003cspan address=\"http://conantlab.org/GenomeVx/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for genome visualization. The GC content of the phage genome was calculated and visualized using GC Content Plotter. The presence of tRNA genes in the genome was assessed using tRNAscan-SE version 2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://trna.ucsc.edu/tRNAscan-SE/\u003c/span\u003e\u003cspan address=\"http://trna.ucsc.edu/tRNAscan-SE/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Phylogenetic analysis was performed using MEGA11 software based on both the terminase large subunit sequence and the whole genome sequence. Comparative genomic analysis with related phages was conducted using DiGAlign (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genome.jp/digalign/upload\u003c/span\u003e\u003cspan address=\"https://www.genome.jp/digalign/upload\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).. Since the therapeutic application of phages requires the absence of virulence factors and antibiotic resistance genes, the genome was screened against the Virulence Factor Database (VFDB) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.mgc.ac.cn/VFs/main.htm\u003c/span\u003e\u003cspan address=\"https://www.mgc.ac.cn/VFs/main.htm\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to detect virulence-associated genes and against ResFinder 4.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cge.food.dtu.dk/services/ResFinder/\u003c/span\u003e\u003cspan address=\"https://cge.food.dtu.dk/services/ResFinder/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to identify potential antimicrobial resistance determinants\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Transmission Electron Microscopy\u003c/h2\u003e \u003cp\u003e10 \u0026micro;L of Poly Ethylene Glycol (PEG)-precipitated phage suspension (~\u0026thinsp;10\u0026sup1;\u0026sup1; PFU/mL) was placed onto a clean Parafilm surface. A 300-mesh carbon-coated copper grid (EMS200-Cu; Electron Microscopy Sciences, USA) was gently positioned over the droplet and allowed to adsorb the sample for 25\u0026ndash;30 min. The grid was then carefully lifted, and excess fluid was removed by gentle blotting with filter paper. For negative staining, the grid was inverted (sample side down) onto a drop of 2% (w/v) aqueous phosphotungstic acid solution (pH 6.8) and incubated for 3 min. Residual stain was blotted off, and the grid was air-dried on filter paper to prevent contact with any wet surface. Once completely dried, the grids were visualised and imaged using a Thermo Scientific\u0026trade; Talos L120C transmission electron microscope (operating at 20\u0026ndash;120 kV) at the All-India Institute of Medical Sciences (AIIMS), New Delhi, India.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Host Adsorption Assay\u003c/h2\u003e \u003cp\u003eHost adsorption assay was performed according to the procedure described by Mizuno et al. with slight modifications [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Briefly, SAKp26.2 phage particles were mixed with 26 clinical isolates of \u003cem\u003eK. pneumoniae\u003c/em\u003e with a multiplicity of Infection (MOI) of 0.1 in different tubes and incubated at 37 \u0026ordm;C in static conditions for 40 minutes. At 5-min intervals, microcentrifuge tubes were removed, centrifuged for 4 min at 12,000x g, and the supernatant was collected to determine unabsorbed phages using the small-drop plaque assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Determination of Phage Life Cycle by One-Step Growth Curve\u003c/h2\u003e \u003cp\u003eA one-step growth curve to determine the latency and burst size of the phage was performed as described by Amarillas et al., with slight modifications [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. 26 clinical isolates of \u003cem\u003eK. pneumoniae\u003c/em\u003e were grown to a mid-exponential phase (OD: 0.6). Then, 0.9 ml of mid-log phase was mixed with 0.1 ml of SAKp26.2 in tube-A, MOI 0.1, and kept for 10 min (according to previously evaluated adsorption time) at 37\u0026deg;C in static condition. After incubation, 0.1 ml of the phage\u0026ndash;bacteria suspension was mixed with 9.9 ml of phosphate-buffered saline (PBS) in tube B, and the mixture was vortexed several times. Then 0.1ml from tube B was aspirated and dispensed into tube C, containing 9.9 ml of Luria-Bertani broth, to prepare the mixture. Tube-C was kept in a static incubator at 37 \u0026ordm;C for 140 min. 0.1 ml of the bacterial-phage suspension was taken at 10 min intervals. At each time interval, the sample taken out was centrifuged at 12,000\u0026times; g for 3 min, and the number of unadsorbed bacteriophage in the supernatant was determined by titration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Assessment of Phage Stability\u003c/h2\u003e \u003cp\u003epH, thermal and solvent stability of the SAKp26.2 were assessed according to the protocol described by Chauhan et al, 2024 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.7.1 pH stability\u003c/b\u003e - The pH of LB broth was adjusted to values ranging from 1 to 14 (pH 1, 3, 5, 7, 8, 9, 10, 12, and 14) using 0.1 N HCl or 1 N NaOH. The phage suspension was then mixed with the pH-adjusted media at a 1:1 ratio to achieve a final concentration of approximately 1 \u0026times; 10⁹ PFU/mL. The mixtures were incubated at 37\u0026deg;C for 1 h, after which phage titers were determined to evaluate stability under varying pH conditions.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.7.2 Thermal stability\u003c/b\u003e - Phage suspensions (~\u0026thinsp;1 \u0026times; 10⁹ PFU/mL in PBS) were exposed to different temperatures (37\u0026deg;C, 55\u0026deg;C, 65\u0026deg;C, 75\u0026deg;C, 85\u0026deg;C, and 100\u0026deg;C) for 1 h. After incubation, residual phage infectivity was determined by plaque assay.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.7.3 Solvent stability -\u003c/b\u003e Phage stability in different solvents was evaluated by mixing equal volumes (1:1) of phage suspension with phosphate-buffered saline (PBS), chloroform, or 0.9% saline, resulting in a final concentration of around 1 \u0026times; 10⁹ PFU/mL. The mixtures were incubated at 37\u0026deg;C for 1 h with shaking at 150 rpm. Following incubation, samples were centrifuged at 12,000 \u0026times; g for 10 min, and the supernatant was collected for phage titer determination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Spot Test Assay:\u003c/h2\u003e \u003cp\u003eThe spot test assay was performed according to the procedure described by Chakraborty et al. (2024) with brief modifications [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The PEG-concentrated phage suspension was serially diluted from 10⁹ PFU/mL to 10 PFU/mL, corresponding to 10⁻\u0026sup1; to 10⁻\u0026sup1;⁰ dilutions. Subsequently, 10 \u0026micro;L of each dilution was spotted onto a lawn of mid-log phase bacterial culture prepared on agar plates. The plates were then incubated at 37\u0026deg;C overnight, and lytic activity was assessed based on the formation of clear zones.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Efficiency of Plating (EOP)\u003c/h2\u003e \u003cp\u003eEOP was determined according to the procedure described in Mirzaei et al, 2015 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] with slight modifications. Based on observations of host range of phage SAKp26.2, 16 clinical isolates of \u003cem\u003eK. pneumoniae\u003c/em\u003e; O3933, P4272, U4317, U4444, U4636, P4725, U5485, B5521, U5877, U6350, U6795, U6875, U7224, U198, O775, and U2150; were selected as they showed a range of susceptibility to SAKp26.2 infection. Overnight cultures of 16 clinical isolates of \u003cem\u003eK. pneumoniae\u003c/em\u003e were prepared in LB broth. Phage suspensions were serially diluted from stock in SM buffer. Each diluted phage suspension was mixed with the corresponding overnight bacterial culture at a ratio of 1:9 (phage: bacteria) resulting in decreasing MOI. The mixture was incubated statically at 37\u0026deg;C for 10 min to allow phage adsorption. Post incubation 20 \u0026micro;L of each phage-bacterial mixture was spotted onto LB agar plates and allowed to air dry. The plates were then closed, inverted, and incubated overnight at 37\u0026deg;C.\u003c/p\u003e \u003cp\u003ePlaques formed on the bacterial lawns were examined to assess the degrees of phage infectivity across the clinical isolates. The number of plaques obtained for each isolate was compared with those formed on the reference host strain U5877, which was used for phage amplification. The assay was performed in triplicate to ensure reproducibility.\u003c/p\u003e \u003cp\u003eTo check corelation between EOP and kill kinetics; overnight cultures of the clinical isolates were co-incubated with bacteriophage SAKp26.2 at a multiplicity of infection (MOI) of 1 in individual wells of a sterile 96-well flat-bottom microtiter plate. The plate was incubated at 37\u0026deg;C for 24 hours in an BioTek Epoch-2 microplate reader (Agilent Technologies, USA), with optical density (OD) measurements recorded at 600 nm at defined time intervals.\u003c/p\u003e \u003cp\u003eEach assay was performed in triplicate. For each clinical isolate, a corresponding control well containing bacterial culture without phage was included to serve as a growth control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Analysis of Antibacterial Efficacy of the Bacteriophage SAKp26.2\u003c/h2\u003e \u003cp\u003eA kill-kinetics study was conducted to evaluate the lytic activity of the phage against its host, \u003cem\u003eK. pneumoniae\u003c/em\u003e clinical isolate U5877. The bacterial culture was grown to the mid-exponential phase and harvested by centrifugation at 6,000 \u0026times; g for 5 minutes. The supernatant was discarded, and the pellet was resuspended in fresh growth medium. The resulting suspension was then aliquoted into microcentrifuge tubes. The bacteriophage was added to the bacterial suspensions at multiplicities of infection (MOIs) of 0.1, 1, 10, and 100. Subsequently, 200 \u0026micro;L of each phage\u0026ndash;bacteria mixture was transferred in triplicate into a 96-well microtiter plate. Bacterial growth kinetics were monitored for 5 hours using a BioTek Epoch-2 microplate reader (Agilent Technologies, USA), with optical density at 600 nm recorded every 20 minutes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Assessment of antibiofilm potential of SAKp26.2 and biofilm fluorescent staining\u003c/h2\u003e \u003cp\u003e \u003cem\u003eIn-vitro\u003c/em\u003e antibiofilm assays were initially performed in microtiter plates following the method described by Chauhan et al, 2012 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] with slight modifications. Overnight cultures of host, \u003cem\u003eK. pneumoniae\u003c/em\u003e clinical isolate U5877, were diluted 1:100 in fresh LB broth and incubated statically at 37\u0026deg;C for 24 h to allow mature biofilm formation. After incubation, planktonic cells were removed, and wells were washed three times with 1X PBS. Phage suspension (200 \u0026micro;L; MOI 1) was added to the established biofilms and incubated at 37\u0026deg;C under static conditions for 4, 8, and 12 h. Control wells treated with 1X PBS. Following treatment, wells were washed thrice with PBS. Biofilm reduction was evaluated by viable cell counts and crystal violet (CV) staining. For CFU determination, biofilms were scraped, resuspended in PBS, serially diluted, and plated on LB agar plates, which were incubated at 37\u0026deg;C. For biofilm biomass quantification, wells were stained with 0.1% CV, incubated at room temperature for 15 min, rinsed with 1X PBS three times, and allowed to dry. The bound dye was resuspended in destaining solution [acetone: ethanol (1:4)] and transferred into a fresh 96-well plate. Absorbance was measured at 595 nm. The experiment was performed in triplicate.\u003c/p\u003e \u003cp\u003eFor biofilm fluorescent staining, a mixture of dyes consisting of SYTO 9 (30 \u0026micro;M final concentration) and propidium iodide (5 \u0026micro;M final concentration) in a 1:6 ratio was prepared in autoclaved distilled water by combining 1.5 \u0026micro;L of each dye stock with 997 \u0026micro;L distilled water. Finally, approximately 50 \u0026micro;L of this dye mixture was added to each well to cover the well surface evenly, and the wells were incubated at room temperature in the dark for 15 minutes. Following incubation, unbound dye was removed, and the wells were washed three times with 1X PBS. Biofilms were observed using an inverted fluorescence microscope (EVOS M5000, ThermoFisher, Massachusetts, USA), in which SYTO 9 stained the cells green, and PI stained the dead cells red. All procedures were carried out under minimal light exposure to avoid dye photobleaching. post-treatment and washing,\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Phage-Antibiotic Combination Treatment-Mediated Modulation of Bacterial Growth Kinetics\u003c/h2\u003e \u003cp\u003eA broth microdilution assay was conducted to evaluate the growth kinetics of MDR \u003cem\u003eK. pneumoniae\u003c/em\u003e clinical isolate U5877 under treatment with varying antibiotic concentrations, different phage multiplicities of infection (MOIs), and phage\u0026ndash;antibiotic combinations. Briefly, 100 \u0026micro;L of LB broth was dispensed into each well of a 96-well microtiter plate. Four experimental conditions were included: broth control, antibiotic alone, phage alone, and antibiotic\u0026ndash;phage combination. Gentamicin was two-fold serially diluted in LB broth, ranging from 1.25 mg/mL to 0.61 \u0026micro;g/mL. Phage suspensions were similarly two-fold serially diluted to achieve MOIs ranging from 100 to 0.048. An equal volume of exponentially growing bacterial culture was added to each well to obtain a final inoculum of 5 \u0026times; 10⁵ CFU/mL. Bacterial growth was monitored for 24 h using a BioTek Epoch-2 microplate reader (Agilent Technologies, USA), with optical density measured at 600 nm at 1-h intervals. The same experimental procedure was performed using ceftazidime in place of gentamicin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 \u003cem\u003eIn-vitro\u003c/em\u003e antibiofilm activity of SAKp26.2 in urinary tract catheter using continuous flow set up\u003c/h2\u003e \u003cp\u003e \u003cem\u003eIn-vitro\u003c/em\u003e efficacy of phage SAKp26.2 against device-associated biofilms of MDR \u003cem\u003eK. pneumoniae\u003c/em\u003e isolate was evaluated using a previously described continuous flow system [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Approximately 10\u0026sup2; bacterial cells in 100 \u0026micro;L were injected through the silicon septum of a totally implantable venous access port (TIVAP) catheter. The bacterial cells were allowed to adhere to the internal surface of the TIVAP for 3 h at 37\u0026deg;C. Following adhesion, the continuous flow system was initiated at a flow rate of 300 \u0026micro;l/min and maintained for 24 h to allow biofilm formation within the catheter lumen. Non-adherent cells and spent media were continuously removed and collected in a waste container. After biofilm development, the TIVAP-associated biofilms were treated with SAKp26.2 for 4 h and 8 h under static conditions by instilling the phage suspension into the catheter lumen. Following treatment, biofilm cells were recovered and viable counts were determined by CFU/mL plating. Untreated TIVAP-associated biofilms served as controls.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Scanning electron microscopy of the urinary tract catheters\u003c/h2\u003e \u003cp\u003eFor visualization of biofilm architecture and phage-mediated disruption, field emission scanning electron microscopy (FESEM) was performed with slight modifications of previously described method [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. After aseptic removal of colonized totally implantable venous access ports (TIVAP) from the flow system, approximately 1 cm segments from the catheter tip were cut, and the septum was carefully dissected from the port using a sterile scalpel. The septum and catheter segments were washed twice with sterile PBS buffer to remove loosely attached debris and subsequently the catheter segments were fixed overnight in 2.5% glutaraldehyde prepared in PBS at 4\u0026deg;C. Following fixation, the samples were washed with PBS and subjected to sequential dehydration using graded ethanol concentrations (10, 20, 30, 40, 50, 60, 70, 80, 90, and 100%) for 10 min each. The dehydrated samples were air-dried and sputter-coated with gold prior to FESEM imaging at USIC University of Delhi south campus with ZEISS Gemini SEM 500.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments in this study were performed with a minimum of three independent biological replicates to ensure reproducibility. Statistical analyses were carried out using GraphPad Prism (version 10.0). Differences between groups were evaluated using an unpaired t-test or one-way analysis of variance (ANOVA), as appropriate. A \u003cem\u003ep\u003c/em\u003e-value of less than 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.1 SAKp26.2 exhibits rapid adsorption, short latent period, high burst size, and stability under physiological conditions\u003c/h2\u003e \u003cp\u003eThe bacteriophage SAKp26.2 was isolated from the sewage water sample collected at Madras Medical Mission Hospital, from where \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e clinical strains were also isolated. The phage formed characteristic bullseye plaques on the lawn of the host \u003cem\u003eK. pneumoniae\u003c/em\u003e U5877. The plaques exhibited a clear, core lysis zone measuring approximately 0.20 cm in diameter, surrounded by a semi-transparent halo \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea\u003cb\u003e).\u003c/b\u003e The presence of a halo suggests depolymerase activity resulting from enzymatic degradation of capsular polysaccharides on the bacterial surface [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. TEM analysis revealed that the phage SAKp26.2 possessed an icosahedral head and a short, non-contractile tail, structural features characteristic of members of the family Autographviridae [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The capsid diameter was approximately 50 nm \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb\u003cb\u003e).\u003c/b\u003e TEM images show the adsorption of SAKp26.2 on host bacterial cells, as well as their lysis \u003cb\u003e(Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-S3).\u003c/b\u003e Phage SAKp26.2 exhibited rapid adsorption to its host strain with nearly 95% of phage particles adsorbed within 5 minutes \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec\u003cb\u003e).\u003c/b\u003e The one-step growth curve indicated a short latent period of around 10 minutes, followed by a rise period of 50 minutes. A secondary latent phase (plateau) was observed thereafter. The calculated burst size was approximately 166 PFU per infected cell, indicating the release of 166 progeny virions upon lysis of a single bacterial cell \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed\u003cb\u003e).\u003c/b\u003e The stability of the phage SAKp26.2 was evaluated under varying pH, temperature, and solvent conditions. A marked reduction in phage titer was observed at extreme acidic and alkaline pH. The phage remained stable at neutral pH, followed by a sharp decline at pH 12, and complete degradation of virion particles occurred at pH 1, 3 and 14 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee\u003cb\u003e).\u003c/b\u003e Thermal stability analysis showed that SAKp26.2 retained infectivity up to 55 \u0026ordm;C. However, a sharp decline in phage titer was recorded at 65 \u0026ordm;C and 85 \u0026ordm;C, followed by complete inactivation at 100 \u0026ordm;C \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef\u003cb\u003e).\u003c/b\u003e Solvent stability assessment indicated that SAKp26.2 remained stable in 0.9% saline and PBS \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.2 SAKp26.2 possesses a\u0026thinsp;~\u0026thinsp;41 kb modular lytic genome and a novel species within \u003cem\u003eAutographviridae\u003c/em\u003e\u003c/h2\u003e \u003cp\u003ePhage SAKp26.2 genomic DNA was successfully isolated as a single high-molecular-weight band (~\u0026thinsp;41 kb) on agarose gel electrophoresis \u003cb\u003e(Fig. S4)\u003c/b\u003e. Whole-genome sequencing using the Illumina platform generated a 41,467 bp linear double-stranded DNA genome \u003cb\u003e(GenBank accession: PX974311)\u003c/b\u003e with 97% assembly coverage and a GC content of 52.99% \u003cb\u003e(Fig. S5)\u003c/b\u003e. A total of 50 coding sequences (CDSs) were predicted \u003cb\u003e(supplementary table 2)\u003c/b\u003e, and no tRNA genes were identified.\u003c/p\u003e \u003cp\u003eGenome annotation using Prokka and RAST, followed by manual validation (BLASTp, InterPro, UniProt), revealed a modular organization typical of the family Autographiviridae. The genome is arranged unidirectionally and comprises distinct functional modules including DNA packaging (terminase small and large subunits), host replication machinery takeover proteins (S-adenosyl-L-methionine hydrolase, serine/threonine kinase, and phage-encoded RNA polymerase), DNA replication and nucleotide metabolism (DNA polymerase, primase/helicase, exonuclease, ligase, HNH endonuclease), structural assembly (major capsid protein, scaffolding protein, head-to-tail connector, tail tubular proteins, and tail fiber), DNA injection/ejectosome components, and a complete lysis cassette (endolysin, class II holins, and Rz-like protein) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. The presence of a single-subunit DNA-dependent RNA polymerase confirms its classification among T7-like \u003cem\u003eAutographiviridae\u003c/em\u003e phages [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNotably, CDS-4 encodes a putative S-adenosyl-L-methionine hydrolase and CDS-5 a serine/threonine kinase, while CDS-26 was flagged by DefenseFinder as a potential counter-anti-phage defense protein, suggesting adaptation to overcome host restriction or intracellular immunity systems. CDS-47 encodes a DNA ejectosome-associated peptidoglycan lytic exotransglycosylase, indicating an enzymatically facilitated DNA translocation mechanism. Approximately one-third of CDSs were annotated as hypothetical proteins, representing potential strain-specific adaptive elements \u003cb\u003e(supplementary table 2)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eLifestyle prediction of SAKp26.2 has marked it as a virulent phage \u003cb\u003e(Fig. S6)\u003c/b\u003e, and comprehensive screening revealed absence of integrase, repressor, excisionase, antibiotic resistance genes, virulence factors, and canonical lysogeny modules, supporting a strictly lytic lifestyle and genomic safety \u003cb\u003e(supplementary table 2 \u0026amp;\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003ePhylogenetic analysis based on whole-proteome comparison and terminase large subunit clustering placed SAKp26.2 within the family \u003cem\u003eAutographiviridae\u003c/em\u003e. Whole-genome phylogenetic analysis identified \u003cem\u003eKlebsiella\u003c/em\u003e phage RSU-F9L as the closest relative \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. Whole-genome comparison revealed 93.30%, nucleotide similarity with \u003cem\u003eKlebsiella\u003c/em\u003e phage RSU-F9L and an average nucleotide identity (ANI) value of 91.35% \u003cb\u003e(Fig. S7)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003ePhylogenetic reconstruction based on the terminase large subunit with the most closely related 20 phages identified \u003cem\u003eKlebsiella\u003c/em\u003e phage PEA128 as the closest relative in the TerL-based tree \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e. ANI analysis with \u003cem\u003eKlebsiella\u003c/em\u003e phage PEA128 showed 91.35% identity, which remains below the 95% species demarcation threshold defined by ICTV \u003cb\u003e(Fig. S8)\u003c/b\u003e. These results collectively confirm that SAKp26.2 represents a novel species within the \u003cem\u003eAutographiviridae\u003c/em\u003e lineage.\u003c/p\u003e \u003cp\u003eVIRIDIC intergenomic similarity and Protein Clusters (PC) based intergenomic distance analyses consistently demonstrated values below the 95% ICTV species demarcation threshold while remaining above the 70% genus cutoff \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed \u003cb\u003e\u0026amp; Fig. S9\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSAKp26.2 (41,467 bp) showed strong similarity with the RSU-F9L genome (~\u0026thinsp;39.4 kb) while maintaining distinct regions of sequence divergence. These differences likely correspond to strain-specific genes or hypothetical proteins that may contribute to host adaptation or functional variation. Overall, the alignment supports the close evolutionary relationship of SAKp26.2 with PEA128 and RSU-F9L while highlighting genomic distinctions consistent with its classification as a novel species.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3 Strain-dependent infection dynamics of SAKp26.2 revealed by spot assay lysis, EOP variability, and diverse killing patterns.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSpot assays with phage SAKp26.2 demonstrated plaque formations at varying dilutions across different clinical isolates \u003cb\u003e(Fig. S10)\u003c/b\u003e. The SAKp26.2 suspension produced plaques on the lawn of the U5877 (primary host) clinical strain of \u003cem\u003eK. pneumoniae\u003c/em\u003e up to 10⁻⁷ dilution, with a surrounding halo around the core plaque, forming a characteristic bull\u0026rsquo;s-eye morphology. We skipped spotting the phage suspension from the 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dilution because the titer was very high, and all the sensitive 16 strains showed a similar lysis pattern. Bull\u0026rsquo;s-eye plaques were observed up to 10⁻⁸ dilution on the lawn of O3933 indicating a strong lysis. On the lawn of U2150, plaques were visible up to 10⁻⁷ dilution of phage suspension, while the halo was observed up to 10⁻\u0026sup3; dilution. In O775, plaques were detected up to 10⁻⁵ dilution, with halo formation observed up to 10⁻\u0026sup2; dilution. For P4272, plaques were observed up to a 10⁻⁸ dilution; however, the phage showed strong lysis up to a 10⁻\u0026sup3; dilution, whereas halo formation was visible up to 10⁻⁵ dilution. Isolate no U198 showed relatively poor phage susceptibility, with plaques detectable only up to 10⁻⁵ dilution and no halo formation. Similarly, in U7224, plaques were observed up to 10⁻⁵ dilution without any halo formation. In U6875, halo rings were visible up to 10⁻\u0026sup3; dilution, while plaques were observed up to 10⁻⁶ dilution. In U6795, halo rings were detected up to 10⁻\u0026sup3; dilution and plaques up to a 10⁻⁷ fold dilution. For U6350, a weak killing activity was observed up to 10⁻⁵ dilution. In B5521, halo rings were present up to 10⁻\u0026sup3; dilution, whereas distinct plaques were detected up to 10⁻⁶ dilution. In U5485, distinct halo rings were observed up to 10⁻\u0026sup3; dilution and plaques up to 10⁻⁷ dilution. Similarly, in P4725, halo rings were visible up to 10⁻\u0026sup3; dilution, while plaques were observed up to 10⁻⁶ dilution. In U4636, bull\u0026rsquo;s-eye plaques were observed up to 10⁻⁷ dilution. In U4444, halo rings were present up to10⁻\u0026sup3; dilution, with plaques detectable up to 10⁻⁶ dilution. Clinical isolate U4317 exhibited least susceptivity against phage SAKp26.2 and showed a few faint and indistinct clearing zones, indicating inefficient killing by the phage.\u003c/p\u003e \u003cp\u003eTo establish a reference host for phage SAKp26.2, the phage was amplified in three clinical isolates; O3933, P4725, and U5877. The amplified phage from three different strains were subsequently subjected to titer enumeration on each of the three clinical isolates to evaluate the lytic activity. When SAKp26.2 was amplified in the O3933 and used to infect the three isolates, plaques were observed up to 10⁻\u0026sup1;\u0026sup1; dilution in O3933. Subsequently in strain P4725, plaques were detected up to 10⁻⁵ dilution, while in U5877 plaques were observed up to 10⁻\u0026sup1;⁰ dilution \u003cb\u003e(Fig. S11)\u003c/b\u003e. Similarly, when the phage was amplified in the P4725 and tested on the same three hosts, plaques were detected up to 10⁻⁷, 10⁻\u0026sup1;⁰, and 10⁻⁶ dilutions in O3933, P4725, and U5877, respectively \u003cb\u003e(Fig. S12)\u003c/b\u003e. In contrast, when SAKp26.2 was amplified in the U5877 clinical isolate and subjected to infect the three isolates, plaques were observed up to 10⁻\u0026sup1;\u0026sup1;, 10⁻\u0026sup1;⁰, and 10⁻\u0026sup1;⁰ dilutions in O3933, P4725, and U5877 indicating a similar degree of phage infection in all the strains \u003cb\u003e(Fig. S13)\u003c/b\u003e. All experiments were performed with three biological and technical replicates. Comparative analysis of the efficiency of SAKp26.2 amplified in different hosts indicated that the phage amplified in the U5877 clinical isolate exhibited the most efficient plaque formation across the tested hosts. Therefore, the clinical isolate U5877 was selected as the reference host for phage SAKp26.2\u003c/p\u003e \u003cp\u003eThe efficiency of plating (EOP) analysis \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e revealed substantial variation in the replication efficiency of phage SAKp26.2 across different clinical isolates relative to the reference host U5877 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Among the tested strains, O3933 exhibited the highest replication efficiency (EOP\u0026thinsp;=\u0026thinsp;12.0), followed by U4636 (EOP\u0026thinsp;=\u0026thinsp;3.5) and U6875 (EOP\u0026thinsp;=\u0026thinsp;2.0), indicating strong phage propagation. Intermediate replication efficiency was observed in several isolates, including U5485 (EOP\u0026thinsp;=\u0026thinsp;0.33), U4444 (EOP\u0026thinsp;=\u0026thinsp;0.32), U6795 (EOP\u0026thinsp;=\u0026thinsp;0.27), U4317 (EOP\u0026thinsp;=\u0026thinsp;0.19), P4725 (EOP\u0026thinsp;=\u0026thinsp;0.15), U2150 (EOP\u0026thinsp;=\u0026thinsp;0.14), and O775 (EOP\u0026thinsp;=\u0026thinsp;0.13). In contrast, U7224 exhibited low replication efficiency (EOP\u0026thinsp;=\u0026thinsp;0.003), despite showing moderate susceptibility in the spot assay \u003cb\u003e(Fig. S10)\u003c/b\u003e. Notably, U198 and U6350, which displayed moderate to weak plaque formation, showed no detectable phage replication (EOP\u0026thinsp;=\u0026thinsp;0). Interestingly, P4272 also exhibited an EOP of 0 despite demonstrating relatively strong lytic activity in the spot assay. Overall, the EOP analysis highlights considerable heterogeneity in the replication efficiency of phage SAKp26.2 across clinical isolates, underscoring the importance of host-dependent variability in phage infectivity and propagation.\u003c/p\u003e \u003cp\u003eThe kill kinetics of phage SAKp26.2 were evaluated against 16 clinical isolates to assess the temporal dynamics of phage-mediated bacterial killing and the emergence of resistance. Monitoring changes in OD\u003csub\u003e600\u003c/sub\u003e over time enabled characterisation of host-specific responses, reflecting variations in phage adsorption, replication efficiency, and resistance development patterns. Resistance emergence pattern: clinical isolates U5877, O3933, P4272, U7224, and U4636 developed resistance at 240, 280, 120, 160, and 140 minutes post-infection, respectively \u003cb\u003e(\u003c/b\u003eFig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eq, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ep, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003em, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. Following the emergence of resistance, the bacterial populations recovered and reached OD\u003csub\u003e600\u003c/sub\u003e levels comparable to the untreated bacterial control. Rise-decline-rise pattern: clinical isolates U2150, O775, U6795, B5521, and P4725 initially showed an increase in OD\u003csub\u003e600\u003c/sub\u003e, similar to the control. Upon initiation of phage killing, a sharp decline was observed, followed by another rise due to resistance emergence. In U2150, the initial increase in O.D. was observed at 40 minutes post-infection and continued until 180 minutes, after which a rapid decline was observed, followed by a rise after 320 minutes \u003cb\u003e(\u003c/b\u003eFig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eo\u003cb\u003e)\u003c/b\u003e. Similarly, O775 showed an initial increase from 40 minutes. It persisted until 180 minutes, after which the OD\u003csub\u003e600\u003c/sub\u003e decreased to ~0.2 until 340 minutes. After 340 minutes, a second rise in O.D. was observed due to the emergence of a resistance subpopulation \u003cb\u003e(\u003c/b\u003eFig.\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003en\u003cb\u003e)\u003c/b\u003e. U6795 demonstrated an initial rise at 40 minutes, followed by a decline at 180 minutes, and a second rise at 280 minutes post-infection \u003cb\u003e(\u003c/b\u003eFig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei\u003cb\u003e)\u003c/b\u003e. B5521 exhibited a similar pattern, with an initial rise at 40 minutes, a decline at 180 minutes, and a second rise at 280 minutes \u003cb\u003e(\u003c/b\u003eFig.\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg\u003cb\u003e)\u003c/b\u003e. In P4725, the first rise occurred at 40 minutes, followed by a decline at 180 minutes and a second rise at 320 minutes post-infection \u003cb\u003e(\u003c/b\u003eFig.\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e. None of these isolates reached the OD\u003csub\u003e600\u003c/sub\u003e levels of the bacterial control. Parallel-growth pattern: clinical isolates U6875, U5485, U4444, and U4317 treated with phage showed growth patterns in which the treated bacteria grew parallel to the untreated control, without any sharp decline. However, the O.D. of phage-treated bacteria never reached the O.D. of the control \u003cb\u003e(\u003c/b\u003eFig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. In all these isolates, resistance to the phage appeared within 40 minutes of infection. Weak infection: clinical isolates U198 and U6350 exhibited growth curves similar to those of the bacterial control, with only minor deviations in OD\u003csub\u003e600\u003c/sub\u003e following phage infection \u003cb\u003e(\u003c/b\u003eFig.\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003el, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4 SAKp26.2 suppresses bacterial growth at different MOIs and dismantles mature biofilm architecture\u003c/h2\u003e \u003cp\u003eThe kill-kinetics assay indicated that the phage SAKp26.2 prevented bacterial growth for up to 5 hours at all tested MOIs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea\u003cb\u003e).\u003c/b\u003e SAKp26.2 showed efficient biofilm reduction post-phage treatment on 24-hour-old biofilm with ~\u0026thinsp;99.0% and ~\u0026thinsp;95.0% reduction in viable cell count within 4 hours and 8 hours of phage treatment, respectively \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb\u003cb\u003e).\u003c/b\u003e In the biofilm biomass quantification by crystal violet staining assay, a significant reduction in OD595 was observed at 4 hours and 8 hours post-phage treatment. A\u0026thinsp;~\u0026thinsp;2-fold decrease in optical density was seen after 8 hours of phage treatment \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec\u003cb\u003e).\u003c/b\u003e Live-dead staining further confirmed the antibiofilm activity of phage SAKp26.2. Fluorescence imaging of untreated biofilms showed a dense population of viable bacterial cells predominantly stained green (SYTO9), indicating intact and metabolically active cells. In contrast, phage-treated biofilms exhibited a marked increase in red fluorescence (propidium iodide), representing membrane-compromised or dead bacterial cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee\u003cb\u003e).\u003c/b\u003e The treated biofilms displayed a significant reduction in green fluorescence along with dispersed clusters of red-stained cells, indicating extensive phage-mediated killing within the biofilm matrix.\u003c/p\u003e \u003cp\u003eThree-dimensional surface reconstruction of the fluorescence images further demonstrated a substantial reduction in biofilm thickness and structural integrity following phage treatment. The untreated biofilm formed a dense and elevated architecture, whereas the phage-treated biofilm appeared flattened with reduced biomass and disrupted surface topology \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg\u003cb\u003e)\u003c/b\u003e. These observations corroborate the CFU reduction and crystal violet assay results, confirming the strong antibiofilm efficacy of phage SAKp26.2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Phage-antibiotic treatment suppresses resistance emergence.\u003c/h2\u003e \u003cp\u003eIn the case of ceftazidime treatment, bacterial resistance, indicated by an increase in OD\u003csub\u003e600\u003c/sub\u003e and visible turbidity, emerged approximately 19 h post-treatment at the highest antibiotic concentration (1.25 mg/mL) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u003cb\u003e).\u003c/b\u003e In contrast, treatment with phage SAKp26.2 alone delayed the emergence of bacterial resistance for up to 6 h across all tested MOIs (100\u0026ndash;0.048) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb\u003cb\u003e).\u003c/b\u003e Notably, the phage\u0026ndash;antibiotic combination demonstrated enhanced efficacy, where the highest antibiotic concentration (1.25 mg/mL) together with the highest phage MOI (100) prevented the development of bacterial resistance for the entire 24 h observation period \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec\u003cb\u003e).\u003c/b\u003e The corresponding kill-kinetics curves are given in the supplementary material \u003cb\u003e(Fig. S14, S15, S16).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e A more pronounced effect of the phage\u0026ndash;antibiotic combination was observed with gentamicin. Bacterial resistance developed within 4 h of treatment across the tested antibiotic concentrations (1.25 mg/mL to 0.61 \u0026micro;g/mL) \u003cb\u003e(\u003c/b\u003eFig. \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. In the combination treatment, sustained inhibition of bacterial resistance was observed for 24 h at three different antibiotic\u0026ndash;phage combinations: 1.25 mg/mL with MOI 100, 0.625 mg/mL with MOI 50, and 0.3125 mg/mL with MOI 25 \u003cb\u003e(\u003c/b\u003eFig. \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee\u003cb\u003e).\u003c/b\u003e The corresponding kill-kinetics curves are given in the supplementary material \u003cb\u003e(Fig. S17, S18)\u003c/b\u003e. Representative microtiter plate wells illustrating the antibiotic-phage combination assay are shown in the supplementary material \u003cb\u003e(Fig. S19)\u003c/b\u003e, where wells with effective phage\u0026ndash;antibiotic combinations (1.25 mg/mL with MOI 100, 0.625 mg/mL with MOI 50, and 0.3125 mg/mL with MOI 25) remain clear (OD\u003csub\u003e600\u003c/sub\u003e \u0026le; 0.225), indicating suppression of bacterial growth and resistance development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Bacteriophage SAKp26.2 reduces biofilm of urinary tract catheter within 4 hours of treatment.\u003c/h2\u003e \u003cp\u003eThe antibiofilm efficacy of phage SAKp26.2 was further evaluated against catheter-associated \u003cem\u003eK. pneumoniae\u003c/em\u003e biofilms using a urinary tract catheter model \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea\u003cb\u003e).\u003c/b\u003e Phage treatment resulted in a significant reduction in the number of viable biofilm-associated bacteria compared with the untreated control \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb\u003cb\u003e).\u003c/b\u003e Approximately a\u0026thinsp;~\u0026thinsp;99% decrease in bacterial survival was observed following 4 h of phage exposure, demonstrating the strong antibiofilm activity of SAKp26.2 against catheter-associated \u003cem\u003eK. pneumoniae\u003c/em\u003e biofilms.\u003c/p\u003e \u003cp\u003eScanning electron microscopy (SEM) analysis of untreated catheter surfaces revealed a dense and well-established biofilm structure composed of numerous rod-shaped bacterial cells embedded within an extracellular polymeric matrix The bacterial cells were tightly adhered to the catheter surface, forming multilayered aggregates characteristic of mature biofilms \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn contrast, phage-treated catheter biofilms exhibited a marked disruption of the biofilm architecture after 4 h of treatment \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed\u003cb\u003e).\u003c/b\u003e The treated surfaces showed a substantial reduction in bacterial cell density with scattered and distorted bacterial cells, indicating extensive phage-mediated lysis and detachment of the biofilm matrix. The structural integrity of the biofilm appeared severely compromised, with only a few residual bacterial cells remaining attached to the catheter surface.\u003c/p\u003e \u003cp\u003eCollectively, these results indicate that phage SAKp26.2 effectively disrupts established catheter-associated biofilms by reducing bacterial viability and dismantling the structural integrity of the biofilm matrix.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe surge in the incidence of multidrug-resistant (MDR) \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e infections, particularly those involving biofilm formation on medical devices, highlights the urgent need to develop novel therapeutic strategies [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In this study, a lytic bacteriophage, SAKp26.2, was isolated and characterised, which showed significant antibacterial and antibiofilm activity against clinical isolates of \u003cem\u003eK. pneumoniae\u003c/em\u003e. The plaque morphology of SAKp26.2 showed distinct bullseye plaques with halos, indicating the presence of a depolymerase enzyme \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. The production of halos by phages is strongly associated with polysaccharide depolymerases, which degrade bacterial capsules and the biofilm matrix, allowing phages to infect biofilm-embedded bacteria [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Moreover, transmission electron microscopy confirmed that SAKp26.2 bears an icosahedral head with a short non-contractile tail \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e, which is characteristic of phages belonging to the Autographiviridae family [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The observed high adsorption efficiency \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e and short latent period \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e are indicative of efficient host attachment and lytic replication efficiency of SAKp26.2 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. One of the major infections caused by \u003cem\u003eK. pneumoniae\u003c/em\u003e is infection of the urinary tract [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], which has an average pH of 6.0 and temperature of 37\u0026deg;C [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. For a phage to be effectively used as a therapeutic agent, it must be stable across a range of pH levels, temperatures, and solvents [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The phage remained stable over a pH range of 5\u0026ndash;10 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e, at temperatures up to 55 \u0026ordm;C \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef\u003cb\u003e)\u003c/b\u003e and in solvents such as 0.9% saline, PBS and chloroform \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGenomic analysis of SAKp26.2 showed that it has a compact linear dsDNA genome of size 41.4 kb \u003cb\u003e(Fig. S4)\u003c/b\u003e with 50 coding sequences organised in functional modules typical of Autographviridae phages \u003cb\u003e(supplementary table 2)\u003c/b\u003e. Importantly, no genes related to lysogeny, antibiotic resistance, or bacterial virulence were found in its genome, indicating its purely virulent nature and, thus, safe for therapeutic use \u003cb\u003e(supplementary table 2 \u0026amp;\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. The presence of genes related to S-adenosyl-L-methionine hydrolase and serine/threonine kinases indicates strategies to bypass bacterial immune systems \u003cb\u003e(supplementary table 2)\u003c/b\u003e, thereby enabling efficient infection of clinical isolates. Comparative genomics of SAKp26.2 revealed that it shares a very high level of similarity with Klebsiella phages RSU-F9L and PEA128 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e. The ANI value of the phage is below the 95% ICTV threshold; therefore, it is confirmed to be a new species of \u003cem\u003eAutographiviridae\u003c/em\u003e \u003cb\u003e(Fig. S8)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eThe infectivity and antibacterial potential of phage SAKp26.2 were assessed by host range determination, efficiency of plating (EOP), and kill kinetics analyses of clinical \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates. The killing dynamics and replication efficiency of the phage SAKp26.2 vary considerably across different clinical strains. The host range analysis showed that SAKp26.2 can infect multiple \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates, as observed by plaque formation across a wide range of phage dilutions. The phage SAKp26.2 produced plaques even at higher dilutions (10⁻⁷\u0026ndash;10⁻⁸) on clinical isolates of \u003cem\u003eK. pneumoniae\u003c/em\u003e, like U5877, O3933, U2150, U4636, U6795, and U5485, indicating strong phage infectivity and efficient propagation in these hosts. Reduced susceptibility and limited phage replication were observed in some isolates, such as U198, U7224, and U6350, along with plaque formation only at lower dilutions. The phage produced no plaque on the isolate U4317, with only faint clearing zones \u003cb\u003e(Fig. S10)\u003c/b\u003e, implying inefficient phage-mediated killing in this isolate [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA characteristic bull\u0026rsquo;s-eye morphology of plaque was observed with a central core lysis zone surrounded by a halo during the host range analysis in several isolates \u003cb\u003e(Fig. S10).\u003c/b\u003e Degradation of capsular polysaccharides of bacterial cells by phage-encoded depolymerases results in halo formation around plaques [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. \u003cem\u003eK. pneumoniae\u003c/em\u003e is known for its thick capsule, which can act as a barrier to phage adsorption [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Therefore, the presence of halos in isolates such as U5877, O3933, U6875, U6795, U5485, and P4725 suggests that SAKp26.2 may harbour enzymatic activity that degrades specific capsular structures, thus resulting in stronger infection [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Conversely, the absence of halos in isolates such as U198 and U7224 \u003cb\u003e(Fig. S10)\u003c/b\u003e may indicate differences in capsular composition or reduced activity of the phage depolymerase against those capsule types.\u003c/p\u003e \u003cp\u003eThe efficiency of plating assay was conducted to quantify replication efficiency across different hosts and to further characterise phage infectivity. U5877 was chosen as the reference host considering the comparative amplification experiments, which showed that phage propagated in this isolate produced the most efficient plaque formation across multiple hosts \u003cb\u003e(S11, S12, S13)\u003c/b\u003e. This observation highlights the effect of the propagation host on phage infectivity, as host-specific factors might affect phage particle maturation, adsorption efficiency, or receptor recognition [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e][\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe EOP analysis showed substantial variable phage replication efficiency among the tested isolates. High EOP values observed in O3933 and U4636 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e indicate that these isolates provide highly favourable conditions for phage replication, in some cases exceeding the efficiency observed in the reference host. Similarly, U6875 also supported efficient phage propagation. In contrast, several isolates including P4725, U5485, U6795, O775, U2150, U4444, and U4317 showed intermediate EOP values \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e, indicating moderate susceptibility to the phage [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. These intermediate levels of replication may reflect partial receptor compatibility or the presence of bacterial defense mechanisms that limit phage proliferation [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Extremely low EOP values in U7224 and the inefficient replication observed in U6350, U198, and P4272 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e further highlight the heterogeneity of phage-host interactions among clinical isolates.\u003c/p\u003e \u003cp\u003eKill kinetics experiments were performed to investigate the interaction dynamics between SAKp26.2 and its bacterial hosts. Post-phage infection, four distinct bacterial growth patterns were observed, reflecting differences in replication efficiency, susceptibility, and the emergence of resistance. In the classical lytic pattern, isolates such as U5877 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eq\u003cb\u003e)\u003c/b\u003e, O3933 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ep\u003cb\u003e)\u003c/b\u003e, P4272 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003em\u003cb\u003e)\u003c/b\u003e, U7224 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek\u003cb\u003e)\u003c/b\u003e, and U4636 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e initially exhibited inhibition by the phage but eventually recovered as resistant populations emerged. The timing of resistance development differed among isolates, potentially due to the variations in mutation rates, receptor variability, or intracellular defence mechanisms. The incomplete recovery observed in O3933 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ep\u003cb\u003e)\u003c/b\u003e indicates that the phage retained partial efficacy even after resistance developed.\u003c/p\u003e \u003cp\u003eThe isolates U2150 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eo\u003cb\u003e)\u003c/b\u003e, O775 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003en\u003cb\u003e)\u003c/b\u003e, U6795 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei\u003cb\u003e)\u003c/b\u003e, B5521 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg\u003cb\u003e)\u003c/b\u003e, and P4725 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e showed a rise\u0026ndash;dip\u0026ndash;rise growth pattern, implying complex associations between bacterial growth and phage replication. The bacterial populations initially increased, then declined due to active phage-mediated lysis, and regrew as resistant or partially resistant cells emerged. Interestingly, these isolates did not reach the optical density of the untreated control, indicating that phage SAKp26.2 continued to exert inhibitory effects on bacterial growth even after regrowth.\u003c/p\u003e \u003cp\u003eIsolates such as U6875 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej\u003cb\u003e)\u003c/b\u003e, U5485 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef\u003cb\u003e)\u003c/b\u003e, U4444 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e, and U4317 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e showed a parallel-rise pattern, in which bacterial growth increased along with the control but remained consistently lower than the control. Resistance emerged rapidly within 40 minutes, suggesting either intrinsic resistance mechanisms or rapid adaptation to phage exposure. Meanwhile, isolates U198 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003el\u003cb\u003e)\u003c/b\u003e and U6350 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh\u003cb\u003e)\u003c/b\u003e showed only minor deviations from the control growth curve, indicating minimal susceptibility to SAKp26.2 infection, consistent with their low plaque formation and inefficient EOP values.\u003c/p\u003e \u003cp\u003eThe combined analyses of host range, efficiency of plating (EOP), and kill kinetics underscore the complex and strain-specific interactions between SAKp26.2 and clinical \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates. Although SAKp26.2 can infect multiple strains and replicate efficiently in certain hosts, differences in infection efficiency and the rapid emergence of resistant populations exhibit significant challenges for single-phage therapeutic applications. These results indicate that, given SAKp26.2's considerable antibacterial potential, its most effective clinical use may involve incorporating it into a phage cocktail or phage-antibiotic combination therapy to expand host coverage and delay resistance emergence.\u003c/p\u003e \u003cp\u003eIn the present study, resistance to ceftazidime emerged approximately 19 hours after treatment (at the highest concentration) and around 3 hours post-treatment at other concentrations \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e, while resistance to gentamicin developed within 4 hours across the tested concentrations \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. These findings demonstrate the robust adaptive capacity of \u003cem\u003eK. pneumoniae\u003c/em\u003e under antibiotic selective pressure, which is enabled by multiple resistance mechanisms and the rapid acquisition of adaptive mutations [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Administration of phage SAKp26.2 alone delayed the development of bacterial resistance for up to 6 hours \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e, suggesting sustained lytic activity against the host strain.\u003c/p\u003e \u003cp\u003eHowever, the most pronounced antibacterial effect was observed when the phage was combined with antibiotics, and several phage-antibiotic combinations suppressed bacterial regrowth and prevented the development of resistance throughout the 24-hour observation period \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e. Phage-antibiotic combinations are increasingly recognised for their capacity to delay or suppress the emergence of resistance, which constitutes a significant advantage of combination therapy. In these systems, antibiotics reduce bacterial growth and impose selective pressure, while bacteriophages infect and lyse susceptible bacterial cells, thereby restricting the expansion of resistant subpopulations [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e][\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The enhanced suppression of resistance observed in combination treatments arises from the complementary mechanisms of phages and antibiotics. Antibiotics inhibit essential cellular processes, whereas bacteriophages replicate within bacterial hosts and induce cell lysis, continuously reducing bacterial population density. This dual selective pressure reduces the probability of the resistant mutants emerging in the population. Furthermore, antibiotic-induced physiological stress can enhance bacterial susceptibility to phage infection, thereby contributing to the suppression of resistant populations [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Notably, gentamicin\u0026ndash;phage combinations exhibited a more substantial inhibition of resistance \u003cb\u003e(Fig. S18)\u003c/b\u003e compared to ceftazidime \u003cb\u003e(Fig. S16).\u003c/b\u003e Aminoglycosides such as gentamicin disrupt protein synthesis by targeting the 30S ribosomal subunit, resulting in bactericidal activity and cellular stress that may enhance phage-mediated killing. Similar effects of phage-antibiotic combinations have also been reported against multidrug-resistant Gram-negative pathogens, including ESBL-producing and carbapenem-resistant \u003cem\u003eEscherichia coli\u003c/em\u003e [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Furthermore, the observed suppression of bacterial regrowth at higher phage multiplicities of infection (MOIs) suggests that increasing phage pressure can further limit the expansion of resistant subpopulations. Higher MOIs increase the likelihood of phage-host encounters, thereby accelerating bacterial killing and reducing the probability that resistant mutants will proliferate. Previous studies have also demonstrated that the extent of phage-antibiotic synergy depends on the stoichiometric relationship between phage particles, bacterial cells, and antibiotic concentration, highlighting the importance of optimizing treatment parameters when evaluating phage-antibiotic combination therapy [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA major finding of this study is the strong antibiofilm activity of SAKp26.2. Biofilm-associated infections represent a major clinical challenge because biofilms provide bacteria with protection against antibiotics and host immune responses. Treatment of established biofilms with SAKp26.2 resulted in substantial reductions in viable cell counts and biofilm biomass \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e. Fluorescent live\u0026ndash;dead imaging \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e further confirmed extensive phage-mediated killing within the biofilm matrix and significant disruption of biofilm architecture. Three-dimensional reconstruction of the biofilm structures revealed marked reductions in thickness and structural complexity following phage treatment \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg\u003cb\u003e)\u003c/b\u003e. These observations are consistent with previous reports showing that phages encoding depolymerases can penetrate and degrade biofilm matrices, thereby enhancing bacterial killing [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe ability of SAKp26.2 to disrupt biofilms was further validated in a catheter-associated biofilm model. SEM analysis demonstrated that untreated catheter surfaces were heavily colonized by multilayered bacterial communities embedded within extracellular polymeric substances \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e. In contrast, phage treatment resulted in substantial disruption of the biofilm structure and a dramatic reduction in viable bacterial cells within only four hours \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. Since catheter-associated infections represent a major source of hospital-acquired infections [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], these findings highlight the potential of SAKp26.2 as a candidate for controlling device-associated \u003cem\u003eK. pneumoniae\u003c/em\u003e biofilms [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn this study, bacteriophage SAKp26.2 was isolated and characterized as a novel lytic phage infecting clinical \u003cem\u003eK. pneumoniae\u003c/em\u003e strains. SAKp26.2 belongs to the family \u003cem\u003eAutographviridae\u003c/em\u003e, which is confirmed by morphological and genomic analyses, and it has a compact modular genome of 41.4 kb. Since the SAKp26.2 genome lacks the genes associated with lysogeny, antibiotic resistance, or virulence, it is suitable for therapeutic applications. Comparative genomic and phylogenetic analysis further supported that SAKp26.2 is a novel species of the \u003cem\u003eAutographviridae\u003c/em\u003e lineage. SAKp26.2 can infect multiple clinical \u003cem\u003eK. pneumoniae\u003c/em\u003e, however, differences between spot assay lysis, EOP value, and kill-kinetics indicate that clear zone formation alone does not necessarily reflect productive phage infection. These findings suggest that to precisely evaluate phage infectivity and replication efficiency, a combination of spot assays and quantitative approaches, such as EOP and kill kinetics, is important. Also, SAKp26.2 exhibited strong antibacterial and antibiofilm activity against \u003cem\u003eK. pneumoniae.\u003c/em\u003e The phage was effective in suppressing bacterial growth, significantly reducing biofilm biomass, and disrupting the architecture of mature biofilms in both microtiter plate and catheter-associated biofilm models. Furthermore, treatments with phage-antibiotic combination enhanced bactericidal activity and delayed the emergence of resistance. Thus, these findings demonstrate that bacteriophage SAKp26.2 possesses several characteristics desirable for a therapeutic phage, including a rapid infection, strong antibiofilm activity, genomic safety, and synergistic potential with antibiotics. These properties of SAKp26.2 make it a promising candidate for the control of MDR \u003cem\u003eK. pneumoniae\u003c/em\u003e infections, particularly those associated with biofilms and medical devices.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e: A. and SC would like to thank DBT for the fellowship support. AC would like to thank ICMR for the funding support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e AC and SC contributed to the study conception and design. Material preparation, data collection and analysis were performed by A., DCS, and AJ. The first draft of the manuscript was written by A., SC and DCS. The manuscript was reviewed by AC and RK. AC contributed supervision to resources, project management and grant acquisition. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eWe gratefully acknowledge the financial support provided by ICMR\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003eThe datasets generated and/or analysed during the current study are available at NCBI Gene Bank with accession No PX974311.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWorld Health Organization. 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Using bacteriophages to reduce formation of catheter-associated biofilms by Staphylococcus epidermidis. \u003cem\u003eAntimicrobial agents and chemotherapy\u003c/em\u003e, \u003cem\u003e50\u003c/em\u003e(4), 1268\u0026ndash;1275. https://doi.org/10.1128/AAC.50.4.1268-1275.2006.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-bacteria","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Bacteria](https://link.springer.com/journal/44351)","snPcode":"44351","submissionUrl":"https://submission.springernature.com/new-submission/44351/3","title":"Discover Bacteria","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Klebsiella pneumoniae, Anti-microbial resistance, bacteriophage, bacterial-biofilm, phage-antibiotic combination, efficiency of plating","lastPublishedDoi":"10.21203/rs.3.rs-9111241/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9111241/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAMR has become a multi-million death crisis primarily driven by carbapenem-resistant Gram-negative infections, and it is projected to worsen sharply by 2050, especially in South Asia, unless major interventions are implemented. One of the major contributors to this burden is \u003cem\u003eKlebsiella pneumonia. \u003c/em\u003eThe WHO has categorised carbapenem-resistant \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e as a critical priority pathogen, and the current prevalence of carbapenem-resistant hypervirulent K. pneumoniae has raised concerns about potential future pandemics. The bacteria can mediate the transfer of resistance from environmental to clinical strains. \u0026nbsp;Moreover, biofilm-associated infections caused by multidrug-resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e exacerbate healthcare challenges, particularly in hospital-acquired infections associated with medical devices such as catheters and implants. Amid escalating antibiotic failures, bacteriophages have re-emerged from the pre-antibiotic era as a new-age therapeutic alternative to combat drug-resistant bacterial infections.\u003c/p\u003e\n\u003cp\u003eWe report SAKp26.2, a novel \u003cem\u003eAutographviridae\u003c/em\u003e phage isolated from hospital sewage, which acts as a potent antibacterial and antibiofilm agent against several clinical, drug-resistant \u003cem\u003eK. pneumoniae\u003c/em\u003estrains. Combination treatment of SAKp26.2 with antibiotics resulted in a significant delay in the emergence of treatment resistance compared to monotherapy, supporting its potential as a phage-antibiotic synergistic therapeutic. The phage has a genome size of 41,282 bp and lacks any virulence or antibiotic resistance genes. SAKp26.2 is a strong depolymerase-producing phage and is equipped with other critical lysis-associated enzymes. A rapid elimination of biomass and bacteria residing in biofilms was achieved, resulting in a 99% reduction within 4 hours. Additionally, our study illustrates an association between efficiency of plating and kill-kinetics performance, reflecting how phage replication efficiency within a host population may influence the epidemiological spread of infection. Notably, the phage also showed significant biofilm clearance from urinary tract catheters, indicating potential biomedical applications. Overall, this study integrates fundamental phage biology with a clinically relevant scenario, bridging the gap between bench and bedside.\u003c/p\u003e","manuscriptTitle":"Genomic and translational characterisation of Autographviridae phage SAKp26.2 for catheter-associated biofilm clearance in drug-resistant Klebsiella pneumoniae","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-13 10:36:45","doi":"10.21203/rs.3.rs-9111241/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-08T10:31:50+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-04T13:40:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-01T17:01:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-28T14:21:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-26T17:28:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"112440143239646633942135443406587293430","date":"2026-04-23T01:43:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"171740935897886373523833780657903731703","date":"2026-04-22T06:16:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"49454985731534213770304013898563817199","date":"2026-04-22T06:16:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"11305785105824319755002000391248482063","date":"2026-04-18T19:29:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"72218401181034281254767038481350712413","date":"2026-04-18T06:54:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-18T04:11:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"61139887765541668601564056915693077341","date":"2026-04-17T10:35:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"76127764989582995843539703281472917175","date":"2026-04-17T09:42:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"145661452995835701817390384712071184845","date":"2026-04-17T07:22:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"40775048854701627481472544240948003873","date":"2026-04-12T08:11:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-07T02:54:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-25T08:11:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-20T11:22:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Bacteria","date":"2026-03-20T10:41:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-bacteria","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Bacteria](https://link.springer.com/journal/44351)","snPcode":"44351","submissionUrl":"https://submission.springernature.com/new-submission/44351/3","title":"Discover Bacteria","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2e7a3ba0-5f29-4f38-a8f7-4eb758671bc0","owner":[],"postedDate":"April 13th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-08T10:31:50+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-04T13:40:15+00:00","index":53,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-01T17:01:05+00:00","index":52,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-08T10:40:14+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-13 10:36:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9111241","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9111241","identity":"rs-9111241","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}