Isolation and characterization of a novel phage AbT1 and evaluating its anti-biofilm activity and antibiotic synergy

Isolation and characterization of a novel phage AbT1 and evaluating its anti-biofilm activity and antibiotic synergy | 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 Isolation and characterization of a novel phage AbT1 and evaluating its anti-biofilm activity and antibiotic synergy Xiaoxiao Li, Wanlian Zhang, Haoyu Li, Jiaxin Liu, Chuanwang Yao, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7644592/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Acinetobacter baumannii represents a formidable multidrug-resistant pathogen in healthcare settings. The increase in antimicrobial resistance had led to renewed interest in phage therapy, an approach based on the natural predatory interactions of phages. In this study, a novel phage, AbT1, specific to A. baumannii ATCC 17978, was isolated and subjected to comprehensive characterization. Phage AbT1 demonstrated considerable stability across a broad range of temperatures and pH values, in addition to exhibiting potent lytic activity against A. baumannii isolates. Genomic analysis indicated that phage AbT1 belonged to the Caudoviricetes class and possessed a double-stranded DNA genome of 53,410 bp, containing 78 open reading frames (ORFs). Among these, 29 ORFs were predicted to encode structural or functional proteins. Furthermore, neutralization of A. baumannii -induced cytotoxicity in host cells was observed following treatment with phage AbT1. This investigation also underscored the potential of phage AbT1 in disrupting biofilms formed by A. baumannii . Notably, compared with a single treatment, the combined use of phage AbT1 and antibiotics consistently enhanced the bactericidal effect. Thus, this study emphasized the therapeutic potential of phage AbT1 and offered valuable insights into the treatment of A. baumannii infections through phage-based approaches. Acinetobacter baumannii Phage AbT1 Biological characterization Anti-biofilms Phage–antibiotic synergy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Acinetobacter baumannii , a Gram-negative opportunistic bacterium, ranked among the most formidable clinical pathogens in global [ 1 ]. This microorganism was pervasive in natural environments and exhibited a pronounced ability to persist in healthcare facilities, resisting desiccation, chemical sanitizers, and detergents, which complicates its eradication [ 2 , 3 ]. These traits contributed to a rising trend in nosocomial transmissions linked to A. baumannii . Furthermore, continuous reports highlighted its escalating potential to acquire resistance to multiple antimicrobial classes, resulting in significant therapeutic challenges, increased patient morbidity and mortality, and frequent treatment failures [ 4 ]. The biofilm-forming capability of multidrug-resistant strains enhanced their survival in germ-killing conditions and increased tolerance to disinfectants, thereby aggravating the control of antibiotic-resistant pathogens [ 5 ]. Infections caused by this bacterium include pneumonia, septicemia, wound infections, urinary tract infections, and other diseases [ 6 , 7 ]. Critically ill immunocompromised patients in ICUs were particularly susceptible to infections by carbapenem-resistant A. baumannii (CRAB) [ 8 ]. During the COVID-19 pandemic, cases of A. baumannii co-infections following SARS-CoV-2 infection were repeatedly documented, with global incidence rates of secondary infections 1% among hospitalized COVID-19 patients [ 9 ]. The World Health Organization (WHO) have indicated CRAB as a critical priority pathogen, underscoring the urgent need for novel antimicrobial strategies to combat these infections. Bacteriophages (phages), which are naturally occurring viruses that co-evolve with bacterial hosts, are experiencing a resurgence as potential therapeutic agents against multidrug-resistant (MDR) bacteria [ 10 ]. The lytic process begins when a phage injects its nucleic acid into a bacterial cell, commandeering the host’s biosynthetic machinery to produce new virions, ultimately leading to cell lysis [ 11 ]. Although phage applications sawed historical using, the advent of antibiotics led to their decline. However, increasing antibiotic resistance and the high specificity of phage-bacterium interactions have renewed interest in phages as tailored alternatives to conventional antibiotics[ 12 , 13 ]. Phage presents several theoretical benefits over antibiotics, including narrow host range, self-amplification at infection sites, and efficient bacteriolytic activity [ 14 ]. Notably, phage targeting A. baumannii demonstrated significant anti-biofilm potential [ 15 ]. Recent developments included the formulation of polyvalent phage cocktails for treating complex infections [ 16 ]. Additionally, combining phage with antibiotics arisen as a promising approach to potentiate antibacterial efficacy through phage-antibiotic synergy (PAS), which may reduce the emergence of resistant clones [ 17 ]. Consequently, phage represented a promising alternative for controlling refractory infections and environmental contamination. The initial steps toward therapeutic used involve the isolation and detailed characterization of strictly lytic phage. Rigorous assessment of safety and in vivo efficacy remained imperative before clinical translation. In this study, we reported the isolation and characterization of a novel lytic phage, designated AbT1, capable of infection A. baumannii ATCC 17978. We determined its biological properties, genomic features, and ability to disrupt biofilms. Furthermore, we evaluated the combined antibacterial effects of phage AbT1 with various antibiotics to assess its potential for development as a topical agent for treating A. baumannii infections Materials and methods Strain and growth conditions The A. baumannii strain ATCC 17978 was utilized in this study. The bacterium was grown aerobically in Luria-Bertani (LB) broth for 24 hours at 37°C. For liquid cultures, individual bacterial colonies grown on LB agar plates were inoculated into 3 mL of sterile LB broth. The bacterial suspension was subsequently incubated at 37°C, shaking at 180 rpm. Phage isolation and purification Sewage water samples were collected from Hainan University, China. A 20 mL aliquot of the sample was centrifuged at 4000 rpm for 5 minutes to remove coarse debris. The supernatant was subsequently filtered through a 0.22 µm membrane to eliminate bacterial. Then, 2 mL of the filtrate was combined with 10 mL of LB broth and 1 mL of a log-phase culture of A. baumannii. The mixture was incubated overnight with shaking at 37°C. The enriched lysate was co-cultured with log-phase host bacteria and plated using the LB soft agar. After 24 hours of incubation at 37°C, well-defined plaques were visible. To obtain a pure phage isolate, three consecutive rounds of plaque purification were carried out. A single, clear plaque was selected from a double-agar plate and suspended in 1 mL of SM buffer [ 18 ]. The pure phage was obtained, and stored at 4°C for subsequent experiments. Transmission electron microscopy (TEM) analysis For morphological observation under TEM, high-titer phage suspensions were processed according to previously described methods [ 19 ]. Specifically, purification phage particles were applied to copper grids and stained with 2% phosphotungstic acid (pH 6.8) for 5 minutes. Images were acquired at different magnifications with a JEM-1200EX TEM instrument (JEOL, Japan). Determination of bacteriophage biological characteristics The multiplicity of infection (MOI) of phage AbT1 was measured using a previously described method [ 20 ]. The MOI was assessed by combining phages with host bacteria at varying ratios, including 1:1000, 1:100, 1:10, 1:1, and 10:1. A control group consisting of host bacterial solution without phage was included. The mixture was subsequently incubated at 37°C for 5 hours to facilitate phage adsorption, replication, and host cell lysis. Following incubation, both phage and bacterial titers were quantitatively assessed using the double-layer agar assay. The optimal MOI was identified as the ratio corresponding to the most released offspring phages. One-step growth curve The one-step growth curve of phage AbT1 were evaluated according to a previously described method. Initially, phage particles were added to log-phase bacterial suspensions at the predetermined optimal MOI and allowed to adsorb during 20 minutes incubation at 37°C. To eliminate non-adsorbed phage, the mixture was centrifuged at 4000 × g for 5 minutes. Aliquots were systematically collected every 20 minutes over a total duration of 240 minutes. Quantification of infectious phage particles were performed by double-layer agar assay, and the burst size was computed as the ratio of the peak phage titer to the initial number of infected bacterial. Stability studies The temperature and pH stability of phage AbT1 were evaluated according to standard method. To determine pH stability, phage suspensions were incubated for 1 hour at 37°C across a pH range of 2 to 12. For temperature stability, samples were exposed to temperatures of 4, 37, 45, 55, 65, and 75°C for 1 hour. Following each treatment, the remaining phage titer was quantified using the double-layer agar method. Sequencing and genome analysis The genomic DNA of phage AbT1 was isolated with a phage DNA extraction kit (OMEGA). DNA fragmentation was amplified by PCR and purified with AMPure XP beads. The processed DNA was delivered to Shanghai Lingen Biotechnology Co., Ltd. for second-generation sequencing. The resulting genome sequence of phage AbT1 was compared against the NCBI Nucleotide BLAST database. Genome annotation and prediction of open reading frames were carried out using the RAST platform ( https://rast.nmpdr.org ) and ORF Finder ( https://www.ncbi.nlm.nih.gov/orffinder ). Identification of tRNA-coding regions was conducted with tRNAscan-SE v2.0.12. Screening for potential antimicrobial resistance genes was performed using the CARD database ( https://card.mcmaster.ca ). Comparative analysis with known virulence factors was carried out via the Virulence Factors of Bacterial Pathogens database ( http://www.mgc.ac.cn/VFs/main.htm ). A similarity heatmap among related phage was generated using VIRIDIC. Phylogenetic analysis was conducted with the neighbor-joining algorithm in MEGA v11, and the resulting tree was visualized with the Proksee online platform ( https://proksee.ca ). Phage AbT1 cytotoxicity assay To evaluate the cytotoxicity of the phage AbT1, a test was performed. The A549 cells (10 5 cell well − 1 ) were inoculated in 96-well plate. Meanwhile, A. baumannii were cultivated in LB overnight at 37℃. The activated bacterial liquid was centrifuged at 4500 rpm for 5 minutes to collect the bacteria, and the bacteria were resuspended in DMEM cell medium containing 2% FBS (OD 600 = 0.01). The phage AbT1 was subjected to a 10-fold gradient dilution (10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 ) with PBS. The pre-treated bacterial solution was added to each well of A549 cells. Phage AbT1 was added to the treatment group, and PBS was added to the positive control group, respectively. Cytotoxicity was detected using the CCK-8 and LDH detection kit. Evaluation of anti-biofilm activity of phage AbT1 The biofilm-forming ability of A. baumannii was assessed using the crystal violet staining [ 21 , 22 ]. Overnight bacterial cultures were diluted 1:100 and aliquoted into a 96-well plate, followed by static incubation at 37°C for 24 hours to facilitate mature biofilm formation. After this period, mature biofilms were treated with phage AbT1. A 100 µL volume of each dilution was dispensed into a polystyrene 96-well plate and incubated under the same conditions. Wells containing only bacterial culture medium were used as negative control. Following incubation, biofilms were washed gently three times with distilled water and subsequently stained with 150 µL of 0.1% crystal violet for 10 minutes. Unbound dye was eliminated by washing twice with distilled water. The adsorbed crystal violet was dissolved using 95% ethanol, and the optical density was performed at 570 nm to evaluate biofilm biomass. Phage-antibiotics synergy The antibacterial activity against A. baumannii was evaluated using phage AbT1 in combination with gentamicin or kanamycin at MOI of 0.01 and 100 in LB medium. Antibiotics were introduced into the liquid culture at concentrations corresponding to 1/4 of the minimum inhibitory concentration (MIC). The experimental design included the following groups: untreated A. baumannii control, A. baumannii treated with phage AbT1 alone, A. baumannii exposed to gentamicin (or kanamycin) alone, and A. baumannii treated with both phage AbT1 and gentamicin (or kanamycin). Bacterial growth was monitored at 30 minutes intervals by determining the optical density at 600 nm (OD₆₀₀). All assays were conducted in three independent replicates. Statistical analysis All statistical analyses of data were performed using SPSS version 26. Graphical representations and statistical parameters were generated with GraphPad Prism version 8.0.1. All experiments were independently repeated three times. Results Phage AbT1 isolation and electron microscopy analysis A phage strain called AbT1 was isolated and purified from sewage wastewater Hainan University China. The phage was isolated using A. baumannii ATCC 17978 as the host strain by double-layer agar method. The plaques of phage AbT1 were transparent, possessed well-defined margins, and measured between 7 and 9 mm in diameter (Figs. 1 A and 1 B). Additionally, Morphological observation showed that phage AbT1 had an icosahedral head (76 ± 3 nm, n = 3) as shown by TEM (Figs. 1 C and 1 D). After, the phage AbT1 was underwent 10-fold serial dilutions, resulting in 6×10 8 PFU/mL. Biological characteristics of phage AbT1 The biological properties of phage AbT1 were characterized through several key assays, including the determination of its MOI, one-step growth curve, temperature stability, and pH tolerance. The data shown that highest phage titer was achieved at a phage-to-bacteria ratio of 1:100, indicating an optimal MOI of 0.01 (Fig. 2 A). Furthermore, the one-step growth curve was established to examine the reproductive cycle of the phage. The results revealed 20 minutes latent period, followed by a 120 minutes lysis period. During this period, the production of virus particles increased significantly until it reached a stable plateau. The average burst size was calculated as 30 plaque-forming units (PFU) per infected bacterial (Fig. 2 B). To further characterize the phage, its stability under varying temperatures and pH conditions were evaluated. Phage AbT1 remained thermally stable following 1 hour incubation at 4°C, 37°C, 45°C, and 55°C, with no reduction in titer observed. In contrast, viral viability markedly declined at 65°C and was almost completely abolished at 75°C (Fig. 2 C). Additionally, phage AbT1 stability was assessed across a pH gradient from 2 to 12. The phage titer remained unchanged over a broad pH range (2ཞ10) after 1 hour of exposure. However, a notable decreasing in activity occurred at pH 11 when challenged against A. baumannii , indicating its suitability for using in acidic to neutral environments (Fig. 2 D). Genome sequence analysis of phage AbT1 The genomic DNA of phage AbT1 was sequenced using the Illumina NovaSeq system to characterize its biological attributes. The Fig. 3 presented the complete circular configuration of its genome. The result indicated a double-stranded DNA structure spanning 53,410 bp with a GC composition of 39.7%. The high GC content is often correlated with enhancing molecular stability, contributing to genomic durability. A total of 78 open reading frames (ORFs) were detected, among which 29 (37.18%) were functionally annotated, while the remaining 49 were designated as hypothetical proteins. ORFs functional predictions results were compiled in Table S1 . No tRNA-coding sequences were identified within the genome, implying comprehensive dependence on host translational mechanisms. Consistent with prevailing taxonomic principles, viruses of the same species typically demonstrated over 95% genomic nucleotide similarity [ 23 ]. BLASTn alignments indicated that phage AbT1 exhibited 94.39% sequence identity with Acinetobacter phage YMC/09/02/B1251. Through analyses utilizing BLASTp, InterProScan, and CDD tools, ORFs were organized into five functional clusters: (a) structural and packaging elements, (b) nucleotide metabolism and regulatory processes, (c) replication and control mechanisms, (d) host lysis components, and (e) proteins of unknown function. Notably, ORF56 and ORF57 were recognized as holin and endolysin, respectively, and its were essential factors which enabled phage to induce bacterial lysis. Phylogenetic analysis of phage AbT1 To identify the closest relatives and taxonomic classification of phage AbT1, phylogeny was performed using whole genome sequence and phage conserved protein. Phylogenetic analysis of the genome revealed that phage AbT1 gathered on a distinct branch from Acinetobacter phage YMC11/11/R3177 and Acinetobacter phage YMC/09/02/B1251 in the phylogenetic tree, indicating a relatively distant genetic relationship between phage AbT1 and these two phages (Figs. 4 A and 4 B). A consistent result was observed in the phylogenetic tree constructed from the whole genome of phage AbT1 (Fig. 4 C). Additionally, Genome similarity analysis performed by VIRIDIC, The Acinetobacter phage YMC/09/02/B1251 (NC_019541) exhibited the highest similarity to phage AbT1, with a score of 57.3%, as shown in the heatmap (Fig. S1 ). The complete genome sequence of phage AbT1 was analyzed using NCBI BLAST, revealing high coverage with Acinetobacter phages YMC11/11/R3177 and YMC/09/02/B1251. Consequently, these two phages along with phage AbT1 were selected for whole-genome alignment using Mauve. The analysis demonstrated poor synteny between phage AbT1 and these phages, with notable gene deletions and insertions observed (Fig. S2). In conclusion, phage AbT1 was classified as the member of Caudoviricetes based on their Electron micrograph and genome analysis. The cell viability results of the cells exposed to the isolated phage To evaluate the application potential of phage AbT1 as an antibacterial agent, we detected the hemolysis rate of red blood cells by phage AbT1. As shown in (Fig. 5 A), compared with the control group (Triton-X-100), phage AbT1 showed lower hemolytic activity at different titers, indicating that phage AbT1 was not toxic to red blood cells. Furthermore, to evaluate the toxicity of phage AbT1 to A. baumannii . The effect of phage on A549 cells was detected by CCK-8 test and Lactate dehydrogenase (LDH) value. The result showed that compared with the control group, when different concentrations of phage AbT1 were exogenous added, which there was a significant promoting effect on the metabolic activity of the cells by CCK-8 assay, indicating that phage AbT1 had no cytotoxicity to A549 cells (Fig. 5 B). Moreover, the effect of phage on infected cells was evaluated by LDH, and the results were shown in (Fig. 5 C). Compared with the untreated group, the 10 8 PFU/mL of phage AbT1 could alleviate the toxicity of A. baumannii on A549 cells, with an inhibition rate of 60%, and in a dose-dependent manner. This result indicated that phage AbT1 inhibited the toxic effect of A. baumannii on cells. Phage AbT1 was efficient in eliminating biofilm of A. baumannii According to the crystal violet staining results, the OD 570 absorbance decreased gradually as the titer of phage AbT1 increasing, suggesting that phage AbT1 effectively reduced biofilm formation in a dose-dependent manner. The results demonstrated that phage AbT1 at varying concentrations achieved inhibition rates exceeding 40% after co-incubation with A. baumannii , with a distinct concentration-dependent effect, indicating phage AbT1 concentrations ≥ 10³ PFU/mL can markedly suppress biofilm formation in A. baumannii (Fig. 6 A). Additionally, the degradation effect of phage AbT1 on established mature biofilms was evaluated. The results revealed that a significant reduction in biofilm biomass in phage-treated groups compared to control group, exhibiting a dose-dependent manner, demonstrating its potent efficacy against preformed mature biofilms (Fig. 6 B). The combined inhibitory effect of phage AbT1 and antibiotics on A. baumannii The efficacy of the combined treatment with phage AbT1 and antibiotics (gentamicin or kanamycin) against A. baumannii was assessed by monitoring the optical density at OD₆₀₀ every 30 minutes over 10 hours period. Bacterial growth curve were compared across four conditions: phage AbT1 alone (MOI = 0.01), antibiotic alone (at 1/4 MIC), the combination of phage AbT1 (MOI = 0.01) and antibiotic, and an untreated control. The results demonstrated continuous growth of the untreated control throughout the experiment. Although limited bacterial regrowth was detected after 10 hours in both the phage-only and antibiotic-only treatment groups, the final biomass in these groups substantially reduced compared to the untreated control. At the 10 hours, cultures treated with the combination of phage AbT1 and either gentamicin or kanamycin exhibited markedly suppressed bacterial growth, which was significantly lower than untreated control, as well as in groups receiving phage AbT1 or antibiotics alone (Figs. 7 A and 7 B). The results confirmed that the combination of phage AbT1 and antibiotics (gentamicin or kanamycin) were synergistic. Discussion Acinetobacter baumannii has increasingly been recognized as a significant opportunistic pathogen responsible for both community and nosocomial infections. Multidrug-resistant strains of this bacterium posed considerable treatment difficulties and were linked to increased rates of disease severity and fatal outcomes [ 24 ]. The worldwide spread of these resistant microorganisms is exacerbating the strain on global healthcare systems. As a result, discovering new antimicrobial solutions that target carbapenem-resistant A. baumannii has become an urgent public health priority. Phage-based therapeutics have gained interest as a viable alternative for combating antibiotic-resistant bacteria, given their specific mechanism of bacterial cell lysis [ 25 ]. Given the increasing incidence of antimicrobial-resistant microbes, phage therapy was increasingly regarded as a promising alternative for treating multidrug-resistant bacterial infections [ 26 ]. However, the clinical implementation of such novel interventions remains challenging. These challenges arise from the limited availability of phage suitable for clinical application including detailed genomic analysis, stability assessments to ensure viability during pharmaceutical processing and as well as a lack of systematic in vitro and in vivo evaluations [ 27 ]. In this study, we aimed to isolate and characterize a phage targeting A. baumannii and to evaluate its antimicrobial activity in vitro . A novel phage, designated AbT1, was successfully isolated from sewage wastewater and exhibited strong lytic activity against A. baumannii ATCC 17978. Phage AbT1 showed a notably short latent period of 30 minutes. Reported latent periods for related Podoviridae and Autographiviridae phages range from 15 to 90 minutes [ 28 ]. Generally, phages with shorter latent periods are associated with higher lytic efficiency [ 29 ]. Consistent with this, phage AbT1 demonstrated both a brief latent period and a substantial burst size compared to previously described phages. Furthermore, phage growth curve are critical indicators for classifying lytic behavior and evaluating therapeutic potential. Environmental conditions significantly affect phage stability, which in turn influences the success of phage therapy. Maintaining structural and functional integrity across a range of temperatures and pH levels are essential for phage viability [ 30 ]. For clinical applications, phages must remain stable throughout processing and administration, whether in solution or other medicinal formulations [ 27 , 31 ]. Our results indicated that phage AbT1 maintained high stability across a broad pH range (pH 2ཞ10). It also exhibited considerable temperature stability, remaining viable at temperatures up to 55°C. Other phages, such as vB_AbaM_ABMM1, remain stable between 4°C and 37°C and within pH 5 to 9 [ 32 ]. These findings confirmed that phage AbT1 was a suitable candidate for therapeutic development. Whole-genome sequencing has become increasingly important in the study of bacterial pathogens, particularly for identification, typing, and predicting antimicrobial resistance and virulence factors [ 21 , 33 ]. Whole-genome sequencing of phage AbT1 revealed that it should be classified as a new member of the Caudoviricetes class. The interaction between phage and host bacteria is initiated by the binding of tail fiber proteins to specific receptors on the bacterial cell surface, a critical determinant of host specificity [ 34 , 35 ]. Transmission electron microscopy showed that phage AbT1 had an icosahedral capsid but no distinct tail structure. This structural profile was consistent with phylogenetic analysis indicating that phage AbT1 shared the highest sequence similarity (57.2%) with Acinetobacter phage YMC/09/02/B1251 (NC_019541). Based on these observations, phage AbT1 was proposed to belong to the group of short-tailed phages. Genomic annotation using BlastX and the RAST server identified 78 predicted open reading frames (ORFs). Among these, 62.82% were annotated as hypothetical proteins, while 37.18% showed homology to genes with known functions. Notably, the genome encodes key lytic enzymes including holin (ORF56) and endolysin (ORF57), which permeabilizes the inner membrane and degrades peptidoglycan without disrupting the native microbiome, respectively. This holin-endolysin system is commonly used by DNA phages to enable progeny virion release [ 36 ]. Moreover, genomic analysis confirmed the absence of tRNA genes in phage AbT1, indicating complete dependence on the host’s translational machinery, and was a trait consistent with other phage genomes [ 37 ]. Additionally, no genes associated with lysogeny were detected, supporting the classification of phage AbT1 as a strictly lytic phage. Biofilms diminish antibiotic efficacy by producing an exopolysaccharide (EPS) matrix protects embedded bacterial cells. In contrast, phages can act as potent antibiofilm agents by degrading the EPS layer and lysing bacterial communities within [ 38 ]. Notably, phage AbT1 demonstrated significant biofilm eradicating activity against A. baumannii isolates. These findings aligned with previous reports by Shahed-Al-Mahmud et al. [ 39 ]. It is essential to assess potential synergy or antagonism between phages and antibiotics before clinical application [ 40 ]. To enable complete eradication of bacterial infections and mitigate phage resistance, we evaluated the combination of phage AbT1 with antibiotics treatment for A. baumannii . Phage AbT1 combined with antibiotics (gentamicin and kanamycin) at 1/4 MIC exhibited synergistic effects against A. baumannii in vitro . The combination significantly suppressed bacterial proliferation, consistent with previous reports such as that of phage vB_AbaSi_W9, which also showed enhanced antibacterial activity in combination with antibiotics, suggesting a promising strategy against carbapenem-resistant A. baumannii [ 41 ]. Such combination approaches offered a potential means to reduce the emergence of bacterial resistance during treatment. In summary, the novel phage AbT1 isolated in this study exhibited strong bactericidal and antibiofilm activities against A. baumannii under in vitro conditions. Moreover, its combination with antibiotics resulted in notable synergistic effects, underscoring its potential for further development as an antimicrobial agent. Conclusion In this work, a novel lytic phage targeting A. baumannii , named AbT1, was identified and purified. The phage AbT1 demonstrated robust stability under varying temperature and pH conditions, and displayed significant endolysin-mediated lytic activity in vitro. Considering its physiological traits, genomic features, and enhancing antibacterial effect in combination with antibiotics, phage AbT1 was emerged as a promising therapeutic candidate for combating infections caused by A. baumannii . Declarations Supplementary Information The online version contains supplementary material available at Acknowledgements we gratefully acknowledge the Shanghai Lingen Technology Co., Ltd. for providing technical support. Author contributions X.L.: Methodology, Investigation, Formal analysis, Writing-review & editing, Writing-original draft. W.Z.: Supervision, Methodology, Validation. H.L.: Investigation, Formal analysis. J.L. C.Y. Y.L. H.W. H.H. Y.D.: Supervision, Methodology. S.S.: Writing-review & editing, Funding acquisition, Formal analysis, Conceptualization. X.C.: Writing-review & editing, Supervision, Investigation, Funding acquisition. All authors reviewed the manuscript. Funding This work was financially supported by the National Natural Science Foundation of China (32300033), Hainan Provincial Natural Science Foundation of China (325RC647) and the Scientific Research Foundation of Hainan University (KYQD(ZR)-23141, KYQD (ZR)-23006). Data Availability The datasets generated and analysed during the current study are available from the corresponding author on reasonable request. Ethical approval This article does not contain any studies with human participants or animals by any of the authors. Conflict of interest The authors declare no conflicts of interest. References Zhang, L., Wang, X., Hua, X., Yu, Y., Leptihn, S., & Loh, B. (2022). Therapeutic evaluation of the Acinetobacter baumannii phage Phab24 for clinical use. 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N., Lin, M. D., Lin, L. C., & Lin, N. T. (2021). Phage φAB6-Borne Depolymerase Combats Acinetobacter baumannii Biofilm Formation and Infection. Antibiotics , 10(3), 279. https://doi.org/10.3390/antibiotics10030279 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 Choi, Y. J., Kim, S., Shin, M., & Kim, J. (2024). Synergistic Antimicrobial Effects of Phage vB_AbaSi_W9 and Antibiotics against Acinetobacter baumannii Infection. Antibiotics , 13(7), 680. https://doi.org/10.3390/antibiotics13070680 Supplementary Files GenomeSequencingData.pdf TableS1.docx FigureS1.tif FigureS2.jpg Cite Share Download PDF Status: Posted Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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1","display":"","copyAsset":false,"role":"figure","size":529452,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological characterization of phage AbT1. (\u003cstrong\u003eA\u003c/strong\u003e) Plaque assay of phage AbT1. (\u003cstrong\u003eB\u003c/strong\u003e) Purification phage AbT1 suspension. (\u003cstrong\u003eC\u003c/strong\u003e) TEM image of phage AbT1; scale bar = 200 nm. (\u003cstrong\u003eD\u003c/strong\u003e) Higher-magnification TEM image of phage AbT1; scale bar = 100 nm.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7644592/v1/9e9ff4e661fc39714b02e75d.png"},{"id":92869522,"identity":"ca4d8fff-543e-45ec-94aa-a169fdf4a6bd","added_by":"auto","created_at":"2025-10-06 13:50:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1092322,"visible":true,"origin":"","legend":"\u003cp\u003eBiological characteristics of phage AbT1. (\u003cstrong\u003eA\u003c/strong\u003e) The MOI experiment of phage AbT1.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eB\u003c/strong\u003e) The one-step growth curve of phage AbT1. (\u003cstrong\u003eC\u003c/strong\u003e) Temperature stability of phage AbT1, after incubation at different temperatures (4℃～75℃) for 1 hour, respectively. (\u003cstrong\u003eD\u003c/strong\u003e) pH stability of phage AbT1, data points were phage titers measured after incubation at different pH (2～12) for 1 hour. All analyses were performed in triplicate.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7644592/v1/e545fc4c9da991502c14957f.png"},{"id":92867951,"identity":"9e07835e-1df0-4dc9-adea-0e6320c739bb","added_by":"auto","created_at":"2025-10-06 13:34:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":266311,"visible":true,"origin":"","legend":"\u003cp\u003eGenomic characterization of phage AbT1. The physical map was scaled in kilobases (kb). The inner rings display GC content (brown) and GC skew (green/purple) across the genome. Genes were color-coded by functional category: red represented lysis-related genes (holin, endolysin); blue corresponded to morphogenesis; orange indicated DNA packaging; green denoted replication and metabolism proteins; and black represents hypothetical proteins.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7644592/v1/726de335ffae08e981748c70.png"},{"id":92867952,"identity":"982bc59d-450d-41f5-b934-5165489cf770","added_by":"auto","created_at":"2025-10-06 13:34:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":360021,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of phage AbT1 and related phages.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eAbT1 large subunit evolutionary tree. (\u003cstrong\u003eB\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003ePhage AbT1 capsid protein evolutionary tree. (\u003cstrong\u003eC\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003ePhage AbT1 Whole Genome Evolutionary Tree.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-7644592/v1/566c525820739eaf46c3cc7b.png"},{"id":92867964,"identity":"26c1bb7a-e9a2-4685-926c-93b53b03e60c","added_by":"auto","created_at":"2025-10-06 13:34:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":186304,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of phage AbT1 on the cytotoxicity of cells.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) determination of erythrocyte hemolysis by phage AbT1. (\u003cstrong\u003eB\u003c/strong\u003e) the viability of phage AbT1 on A549 cells was determined by the CCK-8 assay.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eC\u003c/strong\u003e) the protective effect of phage AbT1 on A549 cells after infection with \u003cem\u003eA. baumannii\u003c/em\u003e was analyzed by LDH assay. Values represent the mean ± SEM (n ≥ 3). Asterisks denoted statistically significant differences versus the control: *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01 and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-7644592/v1/0e53b697bc2d44b96ec1b533.png"},{"id":92867957,"identity":"d2655f9b-f338-4451-acf8-49b37377ab0e","added_by":"auto","created_at":"2025-10-06 13:34:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":133634,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of the antibiofilm activity of phage AbT1. (\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eSuppression of biofilm formation by phage AbT1.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eB\u003c/strong\u003e) Disruption of preformed mature biofilm by phage AbT1. Data are expressed as mean ± SEM from three or more independent experiments. Statistically significant differences compared to the control are indicated as *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-7644592/v1/2bc60c4e86c58733d18339ba.png"},{"id":92870586,"identity":"cff5f011-a02c-4dd5-962c-6013726fc76f","added_by":"auto","created_at":"2025-10-06 13:58:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":176864,"visible":true,"origin":"","legend":"\u003cp\u003eAntibacterial synergy between phage AbT1 and antibiotics against \u003cem\u003eA. baumannii\u003c/em\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) The growth curve of \u003cem\u003eA. baumannii\u003c/em\u003e following treatment with phage AbT1 combined with gentamicin at 1/4 MIC. (\u003cstrong\u003eB\u003c/strong\u003e) The growth curve of \u003cem\u003eA. baumannii\u003c/em\u003e following treatment with phage AbT1 combined with kanamycin at 1/4 MIC. Results were presented as mean values from three biologically independent experiments, with duplicate measurements in each.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-7644592/v1/81fcf9f795ba7605974fe95e.png"},{"id":94987232,"identity":"435e21f1-98df-4041-a0a8-7f7db9f69646","added_by":"auto","created_at":"2025-11-03 07:01:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3766128,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7644592/v1/68a981d3-6a46-42ef-81bc-e94229419b20.pdf"},{"id":92867947,"identity":"15608af4-9260-49cd-a00d-b1488d162ce9","added_by":"auto","created_at":"2025-10-06 13:34:04","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":72479,"visible":true,"origin":"","legend":"","description":"","filename":"GenomeSequencingData.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7644592/v1/0aab0474ebecf990a472955f.pdf"},{"id":92869159,"identity":"514a41ea-7519-4448-845c-12cd666eeda3","added_by":"auto","created_at":"2025-10-06 13:42:04","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":26679,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7644592/v1/61bc6554e1904036385b16e2.docx"},{"id":92869163,"identity":"4b1a287d-8c96-4ac2-a374-aae13a870ca5","added_by":"auto","created_at":"2025-10-06 13:42:04","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1261664,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7644592/v1/2cfd7b251aad8b2390d72786.tif"},{"id":92867956,"identity":"d6795b94-1346-4e12-b083-8b6600856a55","added_by":"auto","created_at":"2025-10-06 13:34:04","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":82830,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7644592/v1/29626b7d0cee26d508796c4d.jpg"}],"financialInterests":"","formattedTitle":"Isolation and characterization of a novel phage AbT1 and evaluating its anti-biofilm activity and antibiotic synergy","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eAcinetobacter baumannii\u003c/em\u003e, a Gram-negative opportunistic bacterium, ranked among the most formidable clinical pathogens in global [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This microorganism was pervasive in natural environments and exhibited a pronounced ability to persist in healthcare facilities, resisting desiccation, chemical sanitizers, and detergents, which complicates its eradication [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These traits contributed to a rising trend in nosocomial transmissions linked to \u003cem\u003eA. baumannii\u003c/em\u003e. Furthermore, continuous reports highlighted its escalating potential to acquire resistance to multiple antimicrobial classes, resulting in significant therapeutic challenges, increased patient morbidity and mortality, and frequent treatment failures [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The biofilm-forming capability of multidrug-resistant strains enhanced their survival in germ-killing conditions and increased tolerance to disinfectants, thereby aggravating the control of antibiotic-resistant pathogens [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Infections caused by this bacterium include pneumonia, septicemia, wound infections, urinary tract infections, and other diseases [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Critically ill immunocompromised patients in ICUs were particularly susceptible to infections by carbapenem-resistant \u003cem\u003eA. baumannii\u003c/em\u003e (CRAB) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. During the COVID-19 pandemic, cases of \u003cem\u003eA. baumannii\u003c/em\u003e co-infections following SARS-CoV-2 infection were repeatedly documented, with global incidence rates of secondary infections 1% among hospitalized COVID-19 patients [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The World Health Organization (WHO) have indicated CRAB as a critical priority pathogen, underscoring the urgent need for novel antimicrobial strategies to combat these infections.\u003c/p\u003e\u003cp\u003eBacteriophages (phages), which are naturally occurring viruses that co-evolve with bacterial hosts, are experiencing a resurgence as potential therapeutic agents against multidrug-resistant (MDR) bacteria [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The lytic process begins when a phage injects its nucleic acid into a bacterial cell, commandeering the host\u0026rsquo;s biosynthetic machinery to produce new virions, ultimately leading to cell lysis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Although phage applications sawed historical using, the advent of antibiotics led to their decline. However, increasing antibiotic resistance and the high specificity of phage-bacterium interactions have renewed interest in phages as tailored alternatives to conventional antibiotics[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Phage presents several theoretical benefits over antibiotics, including narrow host range, self-amplification at infection sites, and efficient bacteriolytic activity [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Notably, phage targeting \u003cem\u003eA. baumannii\u003c/em\u003e demonstrated significant anti-biofilm potential [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Recent developments included the formulation of polyvalent phage cocktails for treating complex infections [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Additionally, combining phage with antibiotics arisen as a promising approach to potentiate antibacterial efficacy through phage-antibiotic synergy (PAS), which may reduce the emergence of resistant clones [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Consequently, phage represented a promising alternative for controlling refractory infections and environmental contamination. The initial steps toward therapeutic used involve the isolation and detailed characterization of strictly lytic phage. Rigorous assessment of safety and in vivo efficacy remained imperative before clinical translation.\u003c/p\u003e\u003cp\u003eIn this study, we reported the isolation and characterization of a novel lytic phage, designated AbT1, capable of infection \u003cem\u003eA. baumannii\u003c/em\u003e ATCC 17978. We determined its biological properties, genomic features, and ability to disrupt biofilms. Furthermore, we evaluated the combined antibacterial effects of phage AbT1 with various antibiotics to assess its potential for development as a topical agent for treating \u003cem\u003eA. baumannii\u003c/em\u003e infections\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStrain and growth conditions\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003eA. baumannii\u003c/em\u003e strain ATCC 17978 was utilized in this study. The bacterium was grown aerobically in Luria-Bertani (LB) broth for 24 hours at 37\u0026deg;C. For liquid cultures, individual bacterial colonies grown on LB agar plates were inoculated into 3 mL of sterile LB broth. The bacterial suspension was subsequently incubated at 37\u0026deg;C, shaking at 180 rpm.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePhage isolation and purification\u003c/h3\u003e\n\u003cp\u003eSewage water samples were collected from Hainan University, China. A 20 mL aliquot of the sample was centrifuged at 4000 rpm for 5 minutes to remove coarse debris. The supernatant was subsequently filtered through a 0.22 \u0026micro;m membrane to eliminate bacterial. Then, 2 mL of the filtrate was combined with 10 mL of LB broth and 1 mL of a log-phase culture of \u003cem\u003eA. baumannii.\u003c/em\u003e The mixture was incubated overnight with shaking at 37\u0026deg;C. The enriched lysate was co-cultured with log-phase host bacteria and plated using the LB soft agar. After 24 hours of incubation at 37\u0026deg;C, well-defined plaques were visible. To obtain a pure phage isolate, three consecutive rounds of plaque purification were carried out. A single, clear plaque was selected from a double-agar plate and suspended in 1 mL of SM buffer [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The pure phage was obtained, and stored at 4\u0026deg;C for subsequent experiments.\u003c/p\u003e\n\u003ch3\u003eTransmission electron microscopy (TEM) analysis\u003c/h3\u003e\n\u003cp\u003eFor morphological observation under TEM, high-titer phage suspensions were processed according to previously described methods [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Specifically, purification phage particles were applied to copper grids and stained with 2% phosphotungstic acid (pH 6.8) for 5 minutes. Images were acquired at different magnifications with a JEM-1200EX TEM instrument (JEOL, Japan).\u003c/p\u003e\n\u003ch3\u003eDetermination of bacteriophage biological characteristics\u003c/h3\u003e\n\u003cp\u003eThe multiplicity of infection (MOI) of phage AbT1 was measured using a previously described method [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The MOI was assessed by combining phages with host bacteria at varying ratios, including 1:1000, 1:100, 1:10, 1:1, and 10:1. A control group consisting of host bacterial solution without phage was included. The mixture was subsequently incubated at 37\u0026deg;C for 5 hours to facilitate phage adsorption, replication, and host cell lysis. Following incubation, both phage and bacterial titers were quantitatively assessed using the double-layer agar assay. The optimal MOI was identified as the ratio corresponding to the most released offspring phages.\u003c/p\u003e\n\u003ch3\u003eOne-step growth curve\u003c/h3\u003e\n\u003cp\u003eThe one-step growth curve of phage AbT1 were evaluated according to a previously described method. Initially, phage particles were added to log-phase bacterial suspensions at the predetermined optimal MOI and allowed to adsorb during 20 minutes incubation at 37\u0026deg;C. To eliminate non-adsorbed phage, the mixture was centrifuged at 4000 \u0026times; g for 5 minutes. Aliquots were systematically collected every 20 minutes over a total duration of 240 minutes. Quantification of infectious phage particles were performed by double-layer agar assay, and the burst size was computed as the ratio of the peak phage titer to the initial number of infected bacterial.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eStability studies\u003c/h2\u003e\u003cp\u003eThe temperature and pH stability of phage AbT1 were evaluated according to standard method. To determine pH stability, phage suspensions were incubated for 1 hour at 37\u0026deg;C across a pH range of 2 to 12. For temperature stability, samples were exposed to temperatures of 4, 37, 45, 55, 65, and 75\u0026deg;C for 1 hour. Following each treatment, the remaining phage titer was quantified using the double-layer agar method.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSequencing and genome analysis\u003c/h3\u003e\n\u003cp\u003eThe genomic DNA of phage AbT1 was isolated with a phage DNA extraction kit (OMEGA). DNA fragmentation was amplified by PCR and purified with AMPure XP beads. The processed DNA was delivered to Shanghai Lingen Biotechnology Co., Ltd. for second-generation sequencing. The resulting genome sequence of phage AbT1 was compared against the NCBI Nucleotide BLAST database. Genome annotation and prediction of open reading frames were carried out using the RAST platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://rast.nmpdr.org\u003c/span\u003e\u003cspan address=\"https://rast.nmpdr.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and ORF Finder (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/orffinder\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/orffinder\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Identification of tRNA-coding regions was conducted with tRNAscan-SE v2.0.12. Screening for potential antimicrobial resistance genes was performed using the CARD database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://card.mcmaster.ca\u003c/span\u003e\u003cspan address=\"https://card.mcmaster.ca\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Comparative analysis with known virulence factors was carried out via the Virulence Factors of Bacterial Pathogens database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.mgc.ac.cn/VFs/main.htm\u003c/span\u003e\u003cspan address=\"http://www.mgc.ac.cn/VFs/main.htm\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). A similarity heatmap among related phage was generated using VIRIDIC. Phylogenetic analysis was conducted with the neighbor-joining algorithm in MEGA v11, and the resulting tree was visualized with the Proksee online platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://proksee.ca\u003c/span\u003e\u003cspan address=\"https://proksee.ca\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003ePhage AbT1 cytotoxicity assay\u003c/h3\u003e\n\u003cp\u003eTo evaluate the cytotoxicity of the phage AbT1, a test was performed. The A549 cells (10\u003csup\u003e5\u003c/sup\u003e cell well\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were inoculated in 96-well plate. Meanwhile, \u003cem\u003eA. baumannii\u003c/em\u003e were cultivated in LB overnight at 37℃. The activated bacterial liquid was centrifuged at 4500 rpm for 5 minutes to collect the bacteria, and the bacteria were resuspended in DMEM cell medium containing 2% FBS (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.01). The phage AbT1 was subjected to a 10-fold gradient dilution (10\u003csup\u003e8\u003c/sup\u003e, 10\u003csup\u003e7\u003c/sup\u003e, 10\u003csup\u003e6\u003c/sup\u003e, 10\u003csup\u003e5\u003c/sup\u003e, 10\u003csup\u003e4\u003c/sup\u003e, 10\u003csup\u003e3\u003c/sup\u003e) with PBS. The pre-treated bacterial solution was added to each well of A549 cells. Phage AbT1 was added to the treatment group, and PBS was added to the positive control group, respectively. Cytotoxicity was detected using the CCK-8 and LDH detection kit.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eEvaluation of anti-biofilm activity of phage AbT1\u003c/h2\u003e\u003cp\u003eThe biofilm-forming ability of \u003cem\u003eA. baumannii\u003c/em\u003e was assessed using the crystal violet staining [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Overnight bacterial cultures were diluted 1:100 and aliquoted into a 96-well plate, followed by static incubation at 37\u0026deg;C for 24 hours to facilitate mature biofilm formation. After this period, mature biofilms were treated with phage AbT1. A 100 \u0026micro;L volume of each dilution was dispensed into a polystyrene 96-well plate and incubated under the same conditions. Wells containing only bacterial culture medium were used as negative control. Following incubation, biofilms were washed gently three times with distilled water and subsequently stained with 150 \u0026micro;L of 0.1% crystal violet for 10 minutes. Unbound dye was eliminated by washing twice with distilled water. The adsorbed crystal violet was dissolved using 95% ethanol, and the optical density was performed at 570 nm to evaluate biofilm biomass.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePhage-antibiotics synergy\u003c/h2\u003e\u003cp\u003eThe antibacterial activity against \u003cem\u003eA. baumannii\u003c/em\u003e was evaluated using phage AbT1 in combination with gentamicin or kanamycin at MOI of 0.01 and 100 in LB medium. Antibiotics were introduced into the liquid culture at concentrations corresponding to 1/4 of the minimum inhibitory concentration (MIC). The experimental design included the following groups: untreated \u003cem\u003eA. baumannii\u003c/em\u003e control, \u003cem\u003eA. baumannii\u003c/em\u003e treated with phage AbT1 alone, \u003cem\u003eA. baumannii\u003c/em\u003e exposed to gentamicin (or kanamycin) alone, and \u003cem\u003eA. baumannii\u003c/em\u003e treated with both phage AbT1 and gentamicin (or kanamycin). Bacterial growth was monitored at 30 minutes intervals by determining the optical density at 600 nm (OD₆₀₀). All assays were conducted in three independent replicates.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses of data were performed using SPSS version 26. Graphical representations and statistical parameters were generated with GraphPad Prism version 8.0.1. All experiments were independently repeated three times.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003ePhage AbT1 isolation and electron microscopy analysis\u003c/h2\u003e\u003cp\u003eA phage strain called AbT1 was isolated and purified from sewage wastewater Hainan University China. The phage was isolated using \u003cem\u003eA. baumannii\u003c/em\u003e ATCC 17978 as the host strain by double-layer agar method. The plaques of phage AbT1 were transparent, possessed well-defined margins, and measured between 7 and 9 mm in diameter (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Additionally, Morphological observation showed that phage AbT1 had an icosahedral head (76\u0026thinsp;\u0026plusmn;\u0026thinsp;3 nm, n\u0026thinsp;=\u0026thinsp;3) as shown by TEM (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). After, the phage AbT1 was underwent 10-fold serial dilutions, resulting in 6\u0026times;10\u003csup\u003e8\u003c/sup\u003e PFU/mL.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eBiological characteristics of phage AbT1\u003c/h2\u003e\u003cp\u003eThe biological properties of phage AbT1 were characterized through several key assays, including the determination of its MOI, one-step growth curve, temperature stability, and pH tolerance. The data shown that highest phage titer was achieved at a phage-to-bacteria ratio of 1:100, indicating an optimal MOI of 0.01 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Furthermore, the one-step growth curve was established to examine the reproductive cycle of the phage. The results revealed 20 minutes latent period, followed by a 120 minutes lysis period. During this period, the production of virus particles increased significantly until it reached a stable plateau. The average burst size was calculated as 30 plaque-forming units (PFU) per infected bacterial (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). To further characterize the phage, its stability under varying temperatures and pH conditions were evaluated. Phage AbT1 remained thermally stable following 1 hour incubation at 4\u0026deg;C, 37\u0026deg;C, 45\u0026deg;C, and 55\u0026deg;C, with no reduction in titer observed. In contrast, viral viability markedly declined at 65\u0026deg;C and was almost completely abolished at 75\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eAdditionally, phage AbT1 stability was assessed across a pH gradient from 2 to 12. The phage titer remained unchanged over a broad pH range (2ཞ10) after 1 hour of exposure. However, a notable decreasing in activity occurred at pH 11 when challenged against \u003cem\u003eA. baumannii\u003c/em\u003e, indicating its suitability for using in acidic to neutral environments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eGenome sequence analysis of phage AbT1\u003c/h2\u003e\u003cp\u003eThe genomic DNA of phage AbT1 was sequenced using the Illumina NovaSeq system to characterize its biological attributes. The Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e presented the complete circular configuration of its genome. The result indicated a double-stranded DNA structure spanning 53,410 bp with a GC composition of 39.7%. The high GC content is often correlated with enhancing molecular stability, contributing to genomic durability. A total of 78 open reading frames (ORFs) were detected, among which 29 (37.18%) were functionally annotated, while the remaining 49 were designated as hypothetical proteins. ORFs functional predictions results were compiled in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eNo tRNA-coding sequences were identified within the genome, implying comprehensive dependence on host translational mechanisms. Consistent with prevailing taxonomic principles, viruses of the same species typically demonstrated over 95% genomic nucleotide similarity [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. BLASTn alignments indicated that phage AbT1 exhibited 94.39% sequence identity with \u003cem\u003eAcinetobacter\u003c/em\u003e phage YMC/09/02/B1251. Through analyses utilizing BLASTp, InterProScan, and CDD tools, ORFs were organized into five functional clusters: (a) structural and packaging elements, (b) nucleotide metabolism and regulatory processes, (c) replication and control mechanisms, (d) host lysis components, and (e) proteins of unknown function. Notably, ORF56 and ORF57 were recognized as holin and endolysin, respectively, and its were essential factors which enabled phage to induce bacterial lysis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003ePhylogenetic analysis of phage AbT1\u003c/h2\u003e\u003cp\u003eTo identify the closest relatives and taxonomic classification of phage AbT1, phylogeny was performed using whole genome sequence and phage conserved protein. Phylogenetic analysis of the genome revealed that phage AbT1 gathered on a distinct branch from \u003cem\u003eAcinetobacter\u003c/em\u003e phage YMC11/11/R3177 and \u003cem\u003eAcinetobacter\u003c/em\u003e phage YMC/09/02/B1251 in the phylogenetic tree, indicating a relatively distant genetic relationship between phage AbT1 and these two phages (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). A consistent result was observed in the phylogenetic tree constructed from the whole genome of phage AbT1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Additionally, Genome similarity analysis performed by VIRIDIC, The \u003cem\u003eAcinetobacter\u003c/em\u003e phage YMC/09/02/B1251 (NC_019541) exhibited the highest similarity to phage AbT1, with a score of 57.3%, as shown in the heatmap (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The complete genome sequence of phage AbT1 was analyzed using NCBI BLAST, revealing high coverage with \u003cem\u003eAcinetobacter\u003c/em\u003e phages YMC11/11/R3177 and YMC/09/02/B1251. Consequently, these two phages along with phage AbT1 were selected for whole-genome alignment using Mauve. The analysis demonstrated poor synteny between phage AbT1 and these phages, with notable gene deletions and insertions observed (Fig. S2). In conclusion, phage AbT1 was classified as the member of \u003cem\u003eCaudoviricetes\u003c/em\u003e based on their Electron micrograph and genome analysis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eThe cell viability results of the cells exposed to the isolated phage\u003c/h2\u003e\u003cp\u003eTo evaluate the application potential of phage AbT1 as an antibacterial agent, we detected the hemolysis rate of red blood cells by phage AbT1. As shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), compared with the control group (Triton-X-100), phage AbT1 showed lower hemolytic activity at different titers, indicating that phage AbT1 was not toxic to red blood cells. Furthermore, to evaluate the toxicity of phage AbT1 to \u003cem\u003eA. baumannii\u003c/em\u003e. The effect of phage on A549 cells was detected by CCK-8 test and Lactate dehydrogenase (LDH) value. The result showed that compared with the control group, when different concentrations of phage AbT1 were exogenous added, which there was a significant promoting effect on the metabolic activity of the cells by CCK-8 assay, indicating that phage AbT1 had no cytotoxicity to A549 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Moreover, the effect of phage on infected cells was evaluated by LDH, and the results were shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Compared with the untreated group, the 10\u003csup\u003e8\u003c/sup\u003e PFU/mL of phage AbT1 could alleviate the toxicity of \u003cem\u003eA. baumannii\u003c/em\u003e on A549 cells, with an inhibition rate of 60%, and in a dose-dependent manner. This result indicated that phage AbT1 inhibited the toxic effect of \u003cem\u003eA. baumannii\u003c/em\u003e on cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhage AbT1 was efficient in eliminating biofilm of\u003c/b\u003e \u003cb\u003eA. baumannii\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAccording to the crystal violet staining results, the OD\u003csub\u003e570\u003c/sub\u003e absorbance decreased gradually as the titer of phage AbT1 increasing, suggesting that phage AbT1 effectively reduced biofilm formation in a dose-dependent manner. The results demonstrated that phage AbT1 at varying concentrations achieved inhibition rates exceeding 40% after co-incubation with \u003cem\u003eA. baumannii\u003c/em\u003e, with a distinct concentration-dependent effect, indicating phage AbT1 concentrations\u0026thinsp;\u0026ge;\u0026thinsp;10\u0026sup3; PFU/mL can markedly suppress biofilm formation in \u003cem\u003eA. baumannii\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Additionally, the degradation effect of phage AbT1 on established mature biofilms was evaluated. The results revealed that a significant reduction in biofilm biomass in phage-treated groups compared to control group, exhibiting a dose-dependent manner, demonstrating its potent efficacy against preformed mature biofilms (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe combined inhibitory effect of phage AbT1 and antibiotics on\u003c/b\u003e \u003cb\u003eA. baumannii\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe efficacy of the combined treatment with phage AbT1 and antibiotics (gentamicin or kanamycin) against \u003cem\u003eA. baumannii\u003c/em\u003e was assessed by monitoring the optical density at OD₆₀₀ every 30 minutes over 10 hours period. Bacterial growth curve were compared across four conditions: phage AbT1 alone (MOI\u0026thinsp;=\u0026thinsp;0.01), antibiotic alone (at 1/4 MIC), the combination of phage AbT1 (MOI\u0026thinsp;=\u0026thinsp;0.01) and antibiotic, and an untreated control. The results demonstrated continuous growth of the untreated control throughout the experiment. Although limited bacterial regrowth was detected after 10 hours in both the phage-only and antibiotic-only treatment groups, the final biomass in these groups substantially reduced compared to the untreated control. At the 10 hours, cultures treated with the combination of phage AbT1 and either gentamicin or kanamycin exhibited markedly suppressed bacterial growth, which was significantly lower than untreated control, as well as in groups receiving phage AbT1 or antibiotics alone (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). The results confirmed that the combination of phage AbT1 and antibiotics (gentamicin or kanamycin) were synergistic.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cem\u003eAcinetobacter baumannii\u003c/em\u003e has increasingly been recognized as a significant opportunistic pathogen responsible for both community and nosocomial infections. Multidrug-resistant strains of this bacterium posed considerable treatment difficulties and were linked to increased rates of disease severity and fatal outcomes [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The worldwide spread of these resistant microorganisms is exacerbating the strain on global healthcare systems. As a result, discovering new antimicrobial solutions that target carbapenem-resistant \u003cem\u003eA. baumannii\u003c/em\u003e has become an urgent public health priority. Phage-based therapeutics have gained interest as a viable alternative for combating antibiotic-resistant bacteria, given their specific mechanism of bacterial cell lysis [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Given the increasing incidence of antimicrobial-resistant microbes, phage therapy was increasingly regarded as a promising alternative for treating multidrug-resistant bacterial infections [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, the clinical implementation of such novel interventions remains challenging. These challenges arise from the limited availability of phage suitable for clinical application including detailed genomic analysis, stability assessments to ensure viability during pharmaceutical processing and as well as a lack of systematic \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e evaluations [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, we aimed to isolate and characterize a phage targeting \u003cem\u003eA. baumannii\u003c/em\u003e and to evaluate its antimicrobial activity \u003cem\u003ein vitro\u003c/em\u003e. A novel phage, designated AbT1, was successfully isolated from sewage wastewater and exhibited strong lytic activity against \u003cem\u003eA. baumannii\u003c/em\u003e ATCC 17978. Phage AbT1 showed a notably short latent period of 30 minutes. Reported latent periods for related \u003cem\u003ePodoviridae\u003c/em\u003e and \u003cem\u003eAutographiviridae\u003c/em\u003e phages range from 15 to 90 minutes [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Generally, phages with shorter latent periods are associated with higher lytic efficiency [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Consistent with this, phage AbT1 demonstrated both a brief latent period and a substantial burst size compared to previously described phages. Furthermore, phage growth curve are critical indicators for classifying lytic behavior and evaluating therapeutic potential. Environmental conditions significantly affect phage stability, which in turn influences the success of phage therapy. Maintaining structural and functional integrity across a range of temperatures and pH levels are essential for phage viability [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. For clinical applications, phages must remain stable throughout processing and administration, whether in solution or other medicinal formulations [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Our results indicated that phage AbT1 maintained high stability across a broad pH range (pH 2ཞ10). It also exhibited considerable temperature stability, remaining viable at temperatures up to 55\u0026deg;C. Other phages, such as vB_AbaM_ABMM1, remain stable between 4\u0026deg;C and 37\u0026deg;C and within pH 5 to 9 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. These findings confirmed that phage AbT1 was a suitable candidate for therapeutic development.\u003c/p\u003e\u003cp\u003eWhole-genome sequencing has become increasingly important in the study of bacterial pathogens, particularly for identification, typing, and predicting antimicrobial resistance and virulence factors [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Whole-genome sequencing of phage AbT1 revealed that it should be classified as a new member of the \u003cem\u003eCaudoviricetes\u003c/em\u003e class. The interaction between phage and host bacteria is initiated by the binding of tail fiber proteins to specific receptors on the bacterial cell surface, a critical determinant of host specificity [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Transmission electron microscopy showed that phage AbT1 had an icosahedral capsid but no distinct tail structure. This structural profile was consistent with phylogenetic analysis indicating that phage AbT1 shared the highest sequence similarity (57.2%) with \u003cem\u003eAcinetobacter\u003c/em\u003e phage YMC/09/02/B1251 (NC_019541). Based on these observations, phage AbT1 was proposed to belong to the group of short-tailed phages. Genomic annotation using BlastX and the RAST server identified 78 predicted open reading frames (ORFs). Among these, 62.82% were annotated as hypothetical proteins, while 37.18% showed homology to genes with known functions. Notably, the genome encodes key lytic enzymes including holin (ORF56) and endolysin (ORF57), which permeabilizes the inner membrane and degrades peptidoglycan without disrupting the native microbiome, respectively. This holin-endolysin system is commonly used by DNA phages to enable progeny virion release [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Moreover, genomic analysis confirmed the absence of tRNA genes in phage AbT1, indicating complete dependence on the host\u0026rsquo;s translational machinery, and was a trait consistent with other phage genomes [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Additionally, no genes associated with lysogeny were detected, supporting the classification of phage AbT1 as a strictly lytic phage.\u003c/p\u003e\u003cp\u003eBiofilms diminish antibiotic efficacy by producing an exopolysaccharide (EPS) matrix protects embedded bacterial cells. In contrast, phages can act as potent antibiofilm agents by degrading the EPS layer and lysing bacterial communities within [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Notably, phage AbT1 demonstrated significant biofilm eradicating activity against \u003cem\u003eA. baumannii\u003c/em\u003e isolates. These findings aligned with previous reports by Shahed-Al-Mahmud et al. [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. It is essential to assess potential synergy or antagonism between phages and antibiotics before clinical application [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. To enable complete eradication of bacterial infections and mitigate phage resistance, we evaluated the combination of phage AbT1 with antibiotics treatment for \u003cem\u003eA. baumannii\u003c/em\u003e. Phage AbT1 combined with antibiotics (gentamicin and kanamycin) at 1/4 MIC exhibited synergistic effects against \u003cem\u003eA. baumannii in vitro\u003c/em\u003e. The combination significantly suppressed bacterial proliferation, consistent with previous reports such as that of phage vB_AbaSi_W9, which also showed enhanced antibacterial activity in combination with antibiotics, suggesting a promising strategy against carbapenem-resistant \u003cem\u003eA. baumannii\u003c/em\u003e [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Such combination approaches offered a potential means to reduce the emergence of bacterial resistance during treatment.\u003c/p\u003e\u003cp\u003eIn summary, the novel phage AbT1 isolated in this study exhibited strong bactericidal and antibiofilm activities against \u003cem\u003eA. baumannii\u003c/em\u003e under \u003cem\u003ein vitro\u003c/em\u003e conditions. Moreover, its combination with antibiotics resulted in notable synergistic effects, underscoring its potential for further development as an antimicrobial agent.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this work, a novel lytic phage targeting \u003cem\u003eA. baumannii\u003c/em\u003e, named AbT1, was identified and purified. The phage AbT1 demonstrated robust stability under varying temperature and pH conditions, and displayed significant endolysin-mediated lytic activity in vitro. Considering its physiological traits, genomic features, and enhancing antibacterial effect in combination with antibiotics, phage AbT1 was emerged as a promising therapeutic candidate for combating infections caused by \u003cem\u003eA. baumannii\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e The online version contains supplementary material available at\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003ewe gratefully acknowledge the Shanghai Lingen Technology Co., Ltd. for providing technical support.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eX.L.: Methodology, Investigation, Formal analysis, Writing-review \u0026amp; editing, Writing-original draft. W.Z.: Supervision, Methodology, Validation. H.L.: Investigation, Formal analysis. J.L. C.Y. Y.L. H.W. H.H. Y.D.: Supervision, Methodology. S.S.: Writing-review \u0026amp; editing, Funding acquisition, Formal analysis, Conceptualization. X.C.: Writing-review \u0026amp; editing, Supervision, Investigation, Funding acquisition. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThis work was financially supported by the National Natural Science Foundation of China (32300033), Hainan Provincial Natural Science Foundation of China (325RC647) and the Scientific Research Foundation of Hainan University (KYQD(ZR)-23141, KYQD (ZR)-23006).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003eThe datasets generated and analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003eThis article does not contain any studies with human participants or animals by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhang, L., Wang, X., Hua, X., Yu, Y., Leptihn, S., \u0026amp; Loh, B. 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Synergistic Antimicrobial Effects of Phage vB_AbaSi_W9 and Antibiotics against \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e Infection. \u003cem\u003eAntibiotics\u003c/em\u003e, 13(7), 680. https://doi.org/10.3390/antibiotics13070680\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Acinetobacter baumannii, Phage AbT1, Biological characterization, Anti-biofilms, Phage–antibiotic synergy","lastPublishedDoi":"10.21203/rs.3.rs-7644592/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7644592/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eAcinetobacter baumannii\u003c/em\u003e represents a formidable multidrug-resistant pathogen in healthcare settings. The increase in antimicrobial resistance had led to renewed interest in phage therapy, an approach based on the natural predatory interactions of phages. In this study, a novel phage, AbT1, specific to \u003cem\u003eA. baumannii\u003c/em\u003e ATCC 17978, was isolated and subjected to comprehensive characterization. Phage AbT1 demonstrated considerable stability across a broad range of temperatures and pH values, in addition to exhibiting potent lytic activity against \u003cem\u003eA. baumannii\u003c/em\u003e isolates. Genomic analysis indicated that phage AbT1 belonged to the \u003cem\u003eCaudoviricetes\u003c/em\u003e class and possessed a double-stranded DNA genome of 53,410 bp, containing 78 open reading frames (ORFs). Among these, 29 ORFs were predicted to encode structural or functional proteins. Furthermore, neutralization of \u003cem\u003eA. baumannii\u003c/em\u003e-induced cytotoxicity in host cells was observed following treatment with phage AbT1. This investigation also underscored the potential of phage AbT1 in disrupting biofilms formed by \u003cem\u003eA. baumannii\u003c/em\u003e. Notably, compared with a single treatment, the combined use of phage AbT1 and antibiotics consistently enhanced the bactericidal effect. Thus, this study emphasized the therapeutic potential of phage AbT1 and offered valuable insights into the treatment of \u003cem\u003eA. baumannii\u003c/em\u003e infections through phage-based approaches.\u003c/p\u003e","manuscriptTitle":"Isolation and characterization of a novel phage AbT1 and evaluating its anti-biofilm activity and antibiotic synergy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-06 13:33:59","doi":"10.21203/rs.3.rs-7644592/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"79845921-4ee3-43ac-9b07-1ed8e252aba4","owner":[],"postedDate":"October 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-31T18:36:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-06 13:33:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7644592","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7644592","identity":"rs-7644592","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}