. 2026 Sep 1;12:202. doi: 10.1038/s41522-026-01137-x

Eradication of catheter-associated MDR Acinetobacter baumannii biofilms mediated by novel phage encoded depolymerase activity

Medhavi Vashisth ^1,², Pawan Kumar ³, Prexha Kapoor ¹, Indu Rani ¹, Priya Sharma ¹, Karan Bhutani ¹, Dheeraj Bansal ⁴, Nitin Virmani ¹, Bidhan Chand Bera ¹, Rajesh Kumar Vaid ¹, Meenakshi Sharma ⁴, Taruna Anand ^1,^✉

PMCID: PMC13658222 PMID: 42864356

Abstract

Bacteriophage-derived depolymerases provide promising scaffolds for the rational design of next-generation anti-biofilm peptides. We report two novel lytic Caudoviricetes phages φAB440 and φAB441, targeting multidrug-resistant hypermucoviscous Acinetobacter baumannii, encoding tail-associated depolymerases with pectate lyase domains and endolysins featuring glycoside hydrolase motifs. Depolymerases and endolysins demonstrated high binding affinity towards capsular polysaccharide K15 (−9.2 kcal/mol) and NAG-NAM tetramers (−7.86 kcal/mol). Synteny analysis revealed significant genome rearrangement and divergence in both phages, more strongly in φAB441. Phage φAB441’s rare codon bias enables efficient depolymerase expression with altered tRNA availability in biofilms. Phages φAB440 and φAB441 suppressed bacterial growth over 24 h at low MOIs (10⁻¹ and 10⁻³), without evidence of resistance development. Four-day-old biofilms formed on urinary catheters were completely eradicated at 10⁹-10⁸ PFU/ml, with ~7.2–7.4 log₁₀ CFU reductions. Phage φAB441, when tested against A. baumannii infection in Caenorhabditis elegans, increased survival of nematodes to 75.97%, 73.97%, and 73.7% at MOIs 1000, 100, and 10, respectively. Our findings highlight the importance of structural validation and in silico analysis towards determining the therapeutic potential of phage lytic proteins.

graphic file with name 41522_2026_1137_Figa_HTML.webp

Subject terms: Antimicrobials, Biofilms, Clinical microbiology, Virology

Introduction

The worldwide dissemination of multidrug-resistant (MDR) strains of Acinetobacter baumannii represents a critical challenge for public health in infectious disease management. Designated as a critical pathogen in the World Health Organization’s Bacterial Priority Pathogens List¹, A. baumannii exhibits extensive resistance mechanisms including production of β-lactamases, over-expression of efflux pump systems, and modification of antimicrobial target sites^2,3. Mainly, this notorious nosocomial pathogen is responsible for ventilator-associated pneumonia (VAP), wound-, burn-, urinary tract- and blood-related infections. Resistant strains of A. baumannii are responsible for 40–67% mortality in cases of VAP and bloodstream-related infections with particularly poor outcomes observed in immunocompromised patient populations^4,5.

These challenges are further compounded by the A. baumannii’s ability to produce recalcitrant biofilms contributing to chronic and persistent infections⁶. Biofilms can develop on both biotic (wounds, tissues, sequestra of bones, teeth etc.) and abiotic surfaces (medical implants and devices, surgical instruments, contact lenses, water pipelines etc.)⁷. Formation of biofilm is a complex, multifactorial process dependent on the growth conditions and cell density⁸. Biofilm development proceeds through a sequential process involving initial reversible attachment mediated by surface adhesins, microcolony formation, pili expression, iron acquisition and subsequent production of an extracellular polymeric substance matrix and final irreversible attachment to the surface^8–10. Mature biofilms are composed of outer extracellular polymeric substance layer, proteins, and extracellular DNA, which collectively forms the matrix that acts as a physical barrier to the external agents^11,12. This complex structural organization leads to lower rates of antibiotic penetration, protection of underlying cells, formation of persisters leading to treatment failures^13,14. Comparative studies have demonstrated that biofilm-associated A. baumannii strains can resist up to 1000-fold greater dose of antibiotics than their planktonic counterparts^15,16.

The escalating crisis of antimicrobial resistance has stimulated a renewed interest over the last decade towards phage therapy as a potential alternative intervention¹⁷. Bacteriophages exhibit superior pharmacological properties compared to conventional antibiotics, demonstrating targeted bacteriolytic specificity, minimal host toxicity, auto-dosing at infection sites, reduced ecological toxicity, accelerated discovery pipelines, diverse formulation adaptability, evolutionarily constrained resistance development, effective biofilm disruption, and mechanistically independent action from existing antibiotics^18–22.

One of the key proteins encoded by bacteriophages is endolysin that specifically degrades the bacterial cell wall by cleaving the peptidoglycan component. But the exogenous application of endolysins against Gram-negative bacteria requires outer membrane permeabilizing agents to access cell wall in Gram-negative bacteria^23–26.

However, depolymerases, found in phage tail structures, act exogenously to degrade bacterial surface exopolysaccharides²⁷. They contain either a glycoside hydrolase domain or a polysaccharide lyase domain, producing characteristic halo formations around the phage plaques (visible on bacterial lawn in double-layer agar) through cleavage of specific glycosidic linkages in poly-β-(1,6)-N-acetylglucosamine matrices^28,29. Structural analyses have revealed that depolymerases have a modular organization with specific carbohydrate-binding domains adjacent to catalytic domains, enabling precise receptor binding and EPS degradation^30,31. A therapeutic approach involving combined application of both endolysins and depolymerases is superior for increased bacterial neutralization and biofilm degradation.

Recent advances in phage genomics have significantly shaped our understanding of A. baumannii targeting phages. A manual search on NCBI database (as of 31 January, 2026) resulted in 391 distinct completely sequenced Acinetobacter phage genome sequences. Phylogenetic analyses reveal a substantial genetic diversity, with the isolates clustered into 14 distinct clades that share ≤50% average nucleotide identity (ANI), indicating multiple independent evolutionary origins. Around 80% of sequenced Acinetobacter phages submitted in NCBI lack comprehensive host-range data and only 12% among them have associated structural proteomic details. This underlines the importance of carrying out comprehensive genomic characterization of phages along with analysis of their lytic profiles, as different phage lineages may possess unique host recognition systems, virulence factors, and therapeutic potential.

In the present investigation, we isolated and characterized two novel virulent bacteriophages, against biofilm-forming MDR A. baumannii strains. The results of this study validate the structure-function relation of phage φAB440 and φAB441 lytic proteins, representing distinct evolutionary lineages of phage depolymerases encoding novel tail spike proteins (TSPs) that demonstrate exceptional degradation of A. baumannii biofilms that predominantly dwell on medical devices such as urinary catheters. These findings not only expand the diversity of therapeutic phages but also provide a platform to design novel and stable peptides suitable for clinical and industrial applications as well.

Results

Characterization of Acinetobacter strains

Amongst the eleven strains of Acinetobacter tested for their antibiotic susceptibility, eight were classified as MDR, which are non-susceptible to ≥1 agent in ≥3 antibiotic classes and two strains as XDR (VTCCBAA1618 and 1619), which are non-susceptible to ≥1 agent in all but ≤2 antibiotic classes³² (Supplementary Table 1). Three strains were categorized as strong biofilm formers (Fo53-5BP, VTCCBAA909 and VTCCBAA1084), one was moderate (VTCCBAA1521), two were weak and rests of the five strains were non-biofilm formers (Supplementary Table 1). On the basis of string test, Fo53-5BP (formed >30 cm long string) was found to be hypermucoviscous strain capable of forming strong biofilms.

Characterization of A. baumannii phages

Two distinct phages were obtained, each against VTCCBAA1521 (φAB440) and Fo53-5BP (φAB441). Phage φAB440 formed 3 mm wide diameter plaques, which were clear and circular with halo region surrounding them. Phage φAB441 formed pin pointed plaques with strong halo region (Fig. 1A, B). The halo region kept on increasing in size for 11 days for φAB440 (Day 1 – 9 mm to Day 11 – 36 mm) and 50 days of observation for φAB441 (Day 1 – 6.5 mm to Day 50 – 77 mm) (Fig. 2, video file). Structural morphology by transmission electron microscopy (TEM) (Fig. 1C, D) revealed that phage φAB440 is a Podovirus and has an icosahedral-shaped head of 56.97 ± 2.14 nm diameter, and a tail of 8.61 ± 0.50 nm length with a thickness of 8.79 ± 0.53 nm. Phage φAB441 is a Myovirus and has an icosahedral-shaped head of 75.54 ± 1.87 nm diameter, and a tail of 110.58 ± 3.99 nm length with a thickness of 28.93 ± 1.83 nm. The bacteriophage φAB441 also has a pronounced base plate and short thick tail fibers.

Fig. 2 — Video file: *Acinetobacter baumannii* phage φAB441 exhibited a remarkable and sustained ability to degrade biofilms produced by a hypermucoviscous strain of *Acinetobacter baumannii*. This image represents a dramatic and continuous increase in halo zone diameter (indicative of polysaccharide degradation) over an unprecedented 50-day incubation period at 37 °C. The halo zone expanded from 6.5 mm at day 1 to 77 mm at day 50. This expanding clearance zone reflects potent and aggressive enzymatic activity, mediated by phage-derived depolymerases, relentlessly degrading the resilient biofilm matrix. Such persistent biofilm eradication underscores exceptional potential of φAB441 to overcome the defense mechanisms of *Acinetobacter* biofilms, offering a powerful alternative to conventional antibiotics in combating chronic, multi drug-resistant infections. In the PDF version of this article, please click anywhere on the figure or caption to play the video in a separate window.

Results from pH sensitivity assay (Fig. 1E, F) revealed that φAB440 was stable at pH values ranging from pH4 to pH10 with maximum stability at pH7 and showed 1.20 log reduction in viable phage titre at pH4. Phage φAB441 was stable even at highly acidic values of pH2 with maximum stability at pH7 with 4.2 and 5.37 log reductions in viable phage titre at pH3 and pH2 as compared to original phage titre in SM buffer at pH 7.2. Temperature sensitivity analysis (Fig. 1G, H) showed that phage φAB440 was stable at temperatures ranging from 4 °C to 65 °C with maximum stability at 25 °C and 1.69 log reduction in phage titre at 65 °C. Beyond 65 °C, the phage lost its viability. Phage φAB441 was stable at wider ranges of temperatures from 4 °C to 80 °C with maximum stability at 4 °C and 1.76 and 4.09 log reductions in phage titre at 70 °C and 80 °C, respectively, as compared to original phage titre at room temperature. Both phages φAB440 and φAB441 were observed to be non- sensitive to chloroform at concentrations up to 20%. One-step growth curve indicated that A. baumannii phage φAB440 had eclipse, latent, and rise periods of 27, 31, and 21 min respectively and a burst size of 80. Phage φAB441 had an eclipse period of 24 min, latent period of ~36 min, rise period of 15 min, and burst size of 111 (Fig. 1I, J). Host range analysis showed that φAB440 was strongly lytic on 2/11 (Fo53-5BP and VTBAA1521) strains and mildly lytic on 1/11 (ATCC19606) and showed no lysis on rest 8/11 strains of Acinetobacter spp. tested. Phage φAB441 was lytic only on its original propagating host i.e. Fo53-5BP, and on rest of the strains it did not show any lysis (Supplementary Fig. 1). Results from EOP studies validated the results of spot assay revealing high productivity of infection of phage φAB440 on two and low on one strain, which were lysed during spot assay. Phage φAB441 showed high productivity on 1/1 lysed strain (Supplementary Table 2).

Genome analysis of A. baumannii phages

Two A. baumannii phages isolated in this study shared no significant similarity to each other. However, phage φAB440 revealed relatedness to many Acinetobacter phage genomes submitted in GenBank, showing highest similarity (coverage: 90%, identity: 93.17%) with vB_AbaP_IME46 (GenBank: MN39529.1) followed by vB_AbaP_ZHSHW (coverage: 88%, identity: 94.37%). The maximum orthologous average nucleotide identity (OrthoANI) was found to be 93.38%. Phage φAB441 had low similarity with other phages in the GenBank database. It only revealed significant similarity with 24 other Acinetobacter phages, one Photobacterium phage, and two Acinetobacter strains, showing maximum similarity with Acinetobacter phage AB5 (coverage: 61%, identity: 95.60%). The maximum OrthoANI for φAB441 was 92.24%.

The circular genome of phage φAB440 (Fig. 3a) had all the genes oriented in the same direction, which are organized as functional gene clusters as indicated by those coding of DNA replication and repair, packaging and for structural proteins. The genome of φAB441 (Fig. 3b) is also organized in functionally distinguished clusters. The genome size of φAB441 (87,975 bp) was more than double the size of φAB440 (41,265 bp). Key features of φAB440 and φAB441 phage genomes are listed in Supplementary Table 3. Phage φAB440 coded for 50 CDS, all of which had AUG (Methionine [M]) as their start codon. Out of these 50 CDS, functions were not identified for 20 and were classified as CDS coding for hypothetical proteins, and rest 30 were functionally annotated on the basis of their sequence homology using automated pipelines RASTtk and Prokka and were manually by BLASTp. Phage φAB441 coded for 165 CDS, of which 142 have AUG (Methionine [M]) as start codon, 12 had GTG (Valine [V]) (genes 19, 53, 59, 81, 84, 90, 101, 102, 106, 121, 124, and 140), and rest 11 had TTG (Leucine [L]) (genes 20, 27, 31, 33, 51, 66, 82, 93, 103, 107, and 164) as alternative start codons. Similar approaches for φAB441 revealed that 31% (51) of the predicted CDS code for functional proteins, and for rest 114 no function could be assigned. InterProScan and CDD database search found 14 & 16 conserved domains for φAB440 (Supplementary Table 4) and 40 & 36 domains for φAB441 (Supplementary Table 5). Figure 3 illustrates circular representation of genomes of phages φAB440 and φAB441 depicting the location and function of some of the important annotated genes. No resistance genes, virulence genes or genes coding for toxins were found in either of the phages. Lack of genes associated with integrase, excisionase, repressor and attachment sites in both the phages suggest their lytic nature. This was also confirmed through use of PhageScope, a tool for determining phage lifestyle. Phages φAB440 and φAB441 were predicted to have 15 and 31 Rho-independent transcription terminators respectively (Supplementary Table 6), that were distributed throughout the phage genome, typically located downstream of coding sequences and operons.

Analysis of key phage proteins

Structures of key phage proteins including tail spike proteins/depolymerases (TSPs) and endolysins were determined using AlphaFold and visualized with PyMOL. Modeled structures were validated using QMEANDisCo scores, ERRAT scores, VERIFY3D scores, PROCHECK analysis, and Ramachandran plot statistics (Supplementary Table 7).

Depolymerase genes

CDS44 of phage φAB440 (862 aa), was predicted to be a TSP with a pectate lyase domain having a catalytic barrel with right handed parallel β-helix (Fig. 4a, in pink cartoon). DoGSiteScorer and PrankWeb identified two binding pockets: a deep elongated cleft at N-terminal (Fig. 4a, Pocket 1) as an anchor domain and another solvent-exposed elongated tunnel at β-helix (Fig. 4a, Pocket 2) that could act as the functionally active site of TSP with depolymerase activity.

CDS62 of φAB441 (100 aa) with one partially opened clamp-like active site (Fig. 4c) was predicted to be an N-terminal domain/anchor rather than a complete TSP due to short length, low secondary structure similarity and significant divergence in structural loops. CDS63 of φAB441 (895 aa), with a deep tunnel like binding pocket (Fig. 4d) was predicted to be a TSP. Despite sharing low sequence similarity with TSP of Acinetobacter phages, a high structural homology with functionally validated polysaccharide lyase (PDB: 7W1E; HHPred: 99.8% probability and with Phyre2: 98.4% confidence) identified it to be a canonical TSP³³.

Genes for host lysis/endolysins

In phage φAB440, CDS46 was predicted to be an endolysin with Glycoside Hydrolase family 19 chitinase domain (GH19) (Fig. 4e, in pink). One active site was predicted (Fig. 4e, as yellow surface) with catalytic residue Glu at 55th position (Glu55, blue surface residue).

In phage φAB441, CDS61 (198 aa) was predicted to be an endolysin with a GH19 domain (Fig. 4f, in pink cartoon) lining aromatic residues Trp74 and Tyr75 for substrate binding. A deep horizontal cleft (Fig. 4f, as yellow surface) was predicted to be an active site containing catalytic residue Glu52 (blue surface residue). Alpha-helices dominated both the endolysins, which are usually found in GH19 family hydrolases³⁴. No CBD domains were detected, marking them as globular endolysins, usually present in bacteriophages infecting Gram-negative bacteria³⁵.

CDS41 of φAB441 was also predicted to be a cell wall hydrolase (CWH) with a functional CWH Sleb domain from Hydrolase 2 superfamily (Fig. 4g, in pink cartoon).

In silico interaction of phage TSPs and endolsyins with bacterial receptors

Molecular docking analysis using Autodock (v4.2.6) revealed that TSPs and endolysins of both Acinetobacter phages φAB440 and φAB441 have varying affinity for exopolymeric substances (EPS) present in the biofilm matrix and peptidoglycan fragments found in the bacterial cell wall. Binding affinities in terms of energy, types of interactions and residues involved in those interactions for TSPs are presented in Table 1 and for endolysins are present in Table 2. TSP of φAB440 had strongest binding affinity with Klebsiella CPS K15, followed by K98, K49, CPS type 6 of Actinobacillus pleuropneumoniae, O-antigen, OMPA and CPS type 7 of A. pleuropneumoniae. However, TSP of φAB441 revealed maximum binding affinity for Klebsiella CPS K98, followed by CPS type 6 of A. pleuropneumoniae, then K49, K15, O-antigen, CPS type 7 of A. pleuropneumoniae and OMPA. Overall, TSP binding with ligands is supported by numerous hydrogen bonds along with other nonbonding interactions. Similarly, endolysin of φAB440 showed maximum binding affinity with NAG-NAM tetramer, followed by NAG, NAM, NAM-L-alanine, NAG-NAM dimer, NAM-L-alanyl-D-isoglutamine and PG. Endolysin of φAB441 showed strongest binding affinity with NAG-NAM tetramer, followed by NAM, NAG, NAM-L-alanine, NAG-NAM dimer, PG and NAM-L-alanyl-D-isoglutamine. Regions of phage TSPs and endolysins involved in ligand binding are presented in Fig. 5. Residues of phage TSPs and endolysins involved in various interactions with different ligands are depicted in 2D schematic diagrams generated by LigPlot+ (Supplementary Figs. 2–5).

Table 1.

Summary of the docking results obtained for interaction of phages TSPs with different ligands

Ligands with PubChem ID used for docking with phage TSPs	TSP from phage	Binding affinity (kcal/mol)	Amino acids with hydrogen bonds	Hydrogen bond length (Å)	Amino acids with hydrophobic interactions	Color represented in binding
Capsular polysaccharide type 6, from Actinobacillus pleuropneumoniae (PubChem CID 148055)	φAB440	−8.2	Asn405 (1), Tyr407 (1), Asn410 (2), Asp461 (2), Glu494 (1), Tyr515 (1), Trp523 (1), Asp551 (1)	2.18, 2.75, 2.85, 3.11, 2.78, 2.79, 3.21, 2.99, 2.93, 3.14	Phe373, Tyr374, Tyr402, Gly404, Ser425, Ser430, Asn431, Ala434, Tyr466, Met519, Trp549	Red
	φAB441	−5.3	Asp260 (1), Lys263 (2), Arg292 (4), Asn293 (2), Asn318 (1), Asn319 (2)	3.18, 2.94, 3.27, 2.77, 2.92, 2.99, 3.07, 2.72, 3.21, 3.01, 3.19, 3.23	Thr264, Ile320	Red
Capsular polysaccharide type 7, from Actinobacillus pleuropneumoniae (PubChem CID 195740)	φAB440	−6.6	Asn405 (1), Tyr407 (2), Ser435 (1), Asn431 (1)	3.17, 3.00, 3.15, 2.93, 2.98	Met365, Phe373, Tyr374, Gly404, Ser430, Ala436, Tyr466, Leu468, Trp523	Blue
	φAB441	−4.1	Asp260 (1), Lys263 (2), Asp265 (4), Arg292 (1)	2.83, 2.94, 3.23, 2.93, 2.96, 2.98, 3.21, 3.16	Gln261, Thr264	Blue
Capsular polysaccharide, Klebsiella K15 (PubChem CID 3083199)	φAB440	−9.2	Asp461 (2), Asn405 (1), Tyr407 (2), Glu328 (1), Arg367 (1), Gln358 (1), Gln401 (1), Asn431 (1), Glu494 (2), Tyr515 (1), Gly404 (1), Asn410 (1)	2.84, 3.12, 2.96, 2.86, 2.88, 3.01, 3.04, 2.95, 3.04, 3.3, 2.70, 3.00, 3.02, 3.14, 2.86	Tyr374, Met365, Tyr402, Trp403, Tyr466, Leu468, Met519, Trp523, Trp549	Magenta
	φAB441	−4.8	Asn258 (1), Asp260 (3), Lys263 (2), Thr264 (1), Thr291 (1), Arg292 (2)	2.93, 2.71, 3.03, 3.13, 2.97, 3.06, 2.82, 3.2, 2.99, 3.18	Asp265, Gln261	Magenta
Capsular polysaccharide K49 (PubChem CID 195668)	φAB440	−8.2	Asn405 (2), Tyr407 (1), Asn431 (2), Asp461 (1), Tyr466 (2), Glu494 (1), Tyr515 (1), Trp523 (1)	2.70, 2.96, 2.84, 3.15, 3.18, 2.83, 2.99, 3.27, 3.18, 2.79, 3.07	Tyr374, Tyr402, Gly404, Ser430, Met519, Glu524	Green
Capsular polysaccharide K49 (PubChem CID 195668)	φAB441	−5.1	Asp260 (2), Gln261 (1), Lys263 (2), Arg292 (3)	3.00, 3.16, 3.18, 2.75, 3.11, 2.90, 3.15, 3.19	Asp265, Thr264	Green
Capsular polysaccharide K98 (PubChem CID 197416)	φAB440	−8.4	Asp461 (1), Asn405 (2), Tyr407 (1), Gly404 (2), Asn431 (1), Tyr466 (1)	2.93, 2.95, 3.15, 2.7, 2.70, 3.29, 3.32, 2.84	Phe373, Tyr374, Tyr402, Ser430, Ser435, Leu468, Trp523	Yellow
Capsular polysaccharide K98 (PubChem CID 197416)	φAB441	−5.4	Asp260 (2), Lys263 (1), Thr264 (1), Arg292 (5), Asn293 (2)	2.71, 2.97, 3.14, 3.08, 2.83, 2.88, 2.93, 3.20, 3.28, 3.28, 3.14	Asp265	Yellow
Escherichia coli serotype O86 O-antigen (PubChem CID 91862843)	φAB440	−7.6	Asn405 (5), Gly404 (1), Gln358 (1), Tyr402 (2), Tyr407 (1), Tyr466 (1), Asn431 (1)	2.58, 2.71, 3.11, 3.17, 3.31, 2.83, 3.01, 2.95, 2.95, 2.32, 3.08, 3.31	Tyr374, Trp403, Ser435, Ser430, His496, Gln401, Trp523	Cyan
	φAB441	−4.6	Asp265 (3), Asp260 (3), Gln261 (1), Lys263 (2), Arg292 (2)	2.92, 2.13, 3.00, 2.31, 3.03, 3.04, 3.06, 3.07, 3.09, 2.97, 3.40	Thr264	Cyan
OMPA outer membrane proteins (PubChem SID 135340124)	φAB440	−7.1	Ser435 (2), Tyr407 (1), Asn410 (2), Gly404 (1)	2.31, 3.02, 3.19, 3.14, 3.14, 2.98	Met365, Phe373, Tyr374, Asn405, Asn431, Ala436, Leu468, Tyr466	Orange
OMPA outer membrane proteins (PubChem SID 135340124)	φAB441	−3.6	Gln261 (1), Phe262 (1)	2.98, 3.34	Thr264, Asn267, Leu275	Orange

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Table 2.

Summary of the docking results obtained for interaction of phage endolsyins with different ligands

Ligands with PubChem ID used for docking with phage Endolysins	Endolysins from phage	Binding affinity (kcal/mol)	Amino acids with hydrogen bonds	Hydrogen bond length (Å)	Amino acids with hydrophobic interactions	Color represented in binding
N-acetylglucosamine (PubChem CID 24139)	φAB440	−3.84	Phe16 (1), Gln24 (3), Arg63 (1)	2.82, 2.50, 2.90, 3.11, 2.91	Lys19, Leu20, Asp21, Gly61, Tyr62	Red
N-acetylglucosamine (PubChem CID 24139)	φAB441	−3.34	Tyr99 (1), Asp127 (1), Leu129 (1), Arg130 (1)	2.95, 2.83, 2.71, 2.93	Lys122, Val126, Phe128	Red
Pentaglycine (PubChem CID 81537)	φAB440	−0.78	Pro67 (1), Lys69 (1)	2.71, 2.83	Leu58, Tyr62, Thr64, Gln66, Val68, Leu118	Green
Pentaglycine (PubChem CID 81537)	φAB441	−2.48	Pro109 (1), Thr112 (1), Asn116 (1)	2.85, 2.93, 3.09	Leu111, Trp153, Phe154, His156, Ile177	Green
NAM-L-alanyl-D-isoglutamine (PubChem CID 451714)	φAB440	−3.14	Lys165 (1), Asp166 (1), Lys167 (3)	3.03, 2.72, 2.80, 3.07, 3.30	Ala168, Glu169, Leu170, Lys173	Blue
NAM-L-alanyl-D-isoglutamine (PubChem CID 451714)	φAB441	−1.45	Ser38 (1), Gln41 (1), Asp151 (1), Lys158 (3)	2.37, 2.47, 2.67, 3.21, 3.33, 3.64	Ile37, Gly36, Met150, Leu159, Ser160	Blue
N-acetylmuramic acid (PubChem CID 5462244)	φAB440	−3.65	Phe16 (1), Lys19 (1), Asp (1), Gln24 (1), Arg63 (1)	2.35, 2, 2.08, 2.54, 3.17	Leu15, Gly17, Leu20, Tyr62	Yellow
N-acetylmuramic acid (PubChem CID 5462244)	φAB441	−3.59	Gly36 (2)	2.59, 2.69	Met1, Ile32, Asn33, Ile37, Ser38, Lys39, Phe198	Yellow
NAM-L-alanine (PubChem CID 10970945)	φAB440	−3.29	Lys165 (1), Lys167 (3)	2.63, 2.53, 2.9, 3.35	Asp166, Ala168, Glu169, Leu170	Magenta
NAM-L-alanine (PubChem CID 10970945)	φAB441	−3.07	Lys67 (1), Gly68 (1), Gly72 (1)	2.73, 3.02, 2.66	Gly64, Lys65, Thr73	Magenta
NAG-NAM dimer (PubChem CID 72210857)	φAB440	−3.23	Lys165 (1), Lys167 (3), Lys165 (1), Lys167 (3)	2.63, 2.53, 2.9, 3.35, 2.63, 2.53, 2.9, 3.35	Gly17, Tyr62	Cyan
NAG-NAM dimer (PubChem CID 72210857)	φAB441	−2.62	Gln110 (1), Thr112 (3), Trp113 (2), Asn116 (1)	2.81, 2.71, 2.85, 3.08, 2.69, 2.77, 2.9	Glu62, Tyr71, Gly88, Leu111, Phe154	Cyan
NAG-NAM tetramer (extracted from PDB ID 4BPA)	φAB440	−7.44	Leu15 (1), Phe16 (1), Lys19 (1), Gln24 (2), Tyr53 (2), Gly61 (1), Arg63 (1)	2.67, 2.37, 2.65, 3.03, 3.03, 2.98, 3.33, 3.09, 3.11	Val91, Gln92, Trp95, Phe136, Ile162, Asn163	Orange
NAG-NAM tetramer (extracted from PDB ID 4BPA)	φAB441	−7.86	Gly64 (1)	2.57	Glu52, Thr53, Ile60, Glu61, Glu62, Tyr63, Lys65, Gln110	Orange

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Fig. 5 — Three-dimensional structures of a CDS44 (TSP) of φAB440 and b CDS63 (TSP) of φAB441 depicting ligand binding with various biofilm matrix components listed in Table 1. The color scheme for ligands used in (a) and (b) is as follows: Red, Capsular polysaccharide type 6, from *A. pleuropneumoniae*; Blue, Capsular polysaccharide type 7, from *A. pleuropneumoniae*; Magenta, Capsular polysaccharide, Klebsiella k15; Green, Capsular polysaccharide K49; Yellow, Capsular polysaccharide K98; Cyan, Escherichia coli serotype O86 O-antigen; Orange, OMPA outer membrane proteins. Ligand binding of c CDS46 (Endolysin) of φAB440 d CDS61 (Endolysin) of φAB441 with various peptidogycan fragments found in bacterial cell wall. The color scheme for ligands used in (c) and (d) is Red, N-acetylglucosamine; Green, Pentaglycine; Blue, NAM-L-alanyl-D-isoglutamine; Yellow, N-acetylmuramic acid; Magenta, NAM-L-alanine; Cyan, NAG-NAM dimer; Orange, NAG-NAM tetramer.

Other than polysaccharide ligands, the TSP of φAB440 showed strongest binding interactions with Acinetobacter baumannii carbapenem-associated outer membrane protein CarO2, followed by Omp33, N-terminal domain of Biofilm-controlling Response Regulator BfmR from A. baumannii, CarO, and CarO1. TSP of φAB441 showed strongest binding interaction with Omp33 followed by, CarO, CarO2, CarO1, and N-terminal domain of BfmR. Endolysin of φAB440 had strongest binding energy with peptidoglycan-associated lipoprotein from A. baumannii 4G4X, followed by 4G4W and 4G4V. For endolysins of φAB441 it was strongest with 4G4W, 4G4V, and 4G4X (Supplementary Table 8 and Supplementary Fig. 6).

Genes for DNA packaging

The terminase complex, which is made up of small (TerS) and large terminase subunits (TerL) is responsible for packing phage DNA into the head. In phage φAB440, both TerS (CDS47) and TerL (CDS48) subunits are present, whereas in φAB441 only TerL (CDS33) is present. A terminase detects the cos/pac sites in phage genomes and uses ATP hydrolysis to form sticky ends³⁶. TerS detects two cos sites and stops packaging by cleaving the DNA at second cos site. TerL detects one pac site, so the phages carrying only TerL packages the DNA in phage head using a progressive heedful packaging where rather than cleaving at later cos site, the phage continue packaging until the capsid is full^37–39. The portal proteins act as a motor-binding platform for the terminase complex, which pumps the phage DNA into the procapsid during packaging ensuring a precise and efficient translocation while preventing DNA leakage⁴⁰. The portal protein in φAB441 is present just downstream of the TerL, promoting synchronized and efficient packaging mechanisms. The TerL-based phylogenetic trees of both the phages represented in Fig. 6a, b reveal that φAB440 is closely related to Acinetobacter phage vB_AbaP_APK48 and φAB441 is closely related to Acinetobacter phage BS46.

Fig. 6 — a Phylogenetic tree of φAB440, and b φAB441 on the basis of terminase large subunit (using MEGA12). Phylogenetic tree of c φAB440, and d φAB441 on the basis of major capsid protein (using MEGA12); e Phylogenetic tree of φAB440 (using VICTOR), and f φAB441 (using VICTOR) phage genomes derived from translated amino acid sequences of conserved proteins.

Major structural genes

Structural genes encompass a major portion of the phage genomes. In φAB440 a single structural gene cassette is located in the region spanning from 21 to 38 kb, and in φAB441 at 15 to 42 kb. The major capsid protein (MCP) is the primary structural component of the phage capsid. Both the phages in the current study form an icosahedral head comprising of numerous copies of MCP. It has a functional role in phage assembly, stability, infection, and evolution^41,42. The major capsid protein of φAB440 (CDS36) is 341 aa long and that of φAB441 (CDS37) is 358 aa long. The MCP of φAB441 belongs to phage_cap_E superfamily (Pfam03864). The domains in the phage MCPs evolve slowly and retain ancestral sequence signatures, making them useful for studying evolutionary relationships. MCP-based phylogenetic trees of both the phages represented in Fig. 6c, d reveal that φAB440 is closely related to Acinetobacter phage f70_k44 and φAB441is closely related to Acinetobacter phage vB_AbaM_B9.

Evolutionary relatedness of phages

Phylogenetic analysis of Acinetobacter phages reveals different clustering patterns and evolutionary relationships. The phylogenetic tree of φAB440 inferred from translated protein sequences using the VICTOR D0 distance formula resolved 37 Acinetobacter and other phages (Enterobacteria phage HK620, Vibrio phage vB VhaP VH 5, and an unclassified Friunavirus sp. BRC001) with an average pseudo-bootstrap support of 21% (Fig. 6e, f). These phages were selected based on their taxonomic relations with the φAB440 (from genus Friunavirus in subfamily Beijerinckvirinae of Autographiviridae) and phage HK620 was selected as it encodes structural homologue of φAB440 tail spike protein. Phage φAB440 was clustered with phages from the “KEN Cluster” (65% bootstrap), which includes clinically significant phages (v_Ab_164_KEN_01-03), Acinetobacter phages AB_SZ6 and vB_AbaP APK14. A total 27 species-level clusters with high genetic diversity were identified and 2 genus-level clusters were also identified that separated Acinetobacter phages from Enterobacteria and Vibrio outliers. The phylogenetic classification for φAB441 diversified 15 Acinetobacter phages and 4 other unclassified Caudoviricetes in 2 major clades and outliers including (Pseudomonas, Aeromonas, Escherichia, Geobacillus phages and φAB440) with high support (~76%). Phage φAB441 shared a monophyletic clade with TaPaz, vB_AbaM_B9, Phab24, and BS46, and recently accessioned phages Ab7, Ab5, Ab3-TPeters suggesting common ancestry. Within this clade, φAB441 showed maximum similarity with vB_AbaM_B9, and TaPaz. The same similarity pattern was observed in calculating AAI values using TerL and MCP for φAB441 validating the placement. Phages φAB440 and φAB441 though isolated from geographically close locations did not cluster together, indicating divergent evolution within a local geographical niche. Pseudomonas phage PAE2 and Aeromonas phage VTCCBPA5 (a previous phage isolate from the same laboratory) branched outside the Acinetobacter group, consistent with host genus divergence despite sharing similar geographical niche. Escherichia phage Cba120 and Geobacillus phage GBK1 (thermotolerant) also served as distant outliers, validating tree topology. A total of 17 species-level, and 7 genus-level clusters were classified, aligning with phylogenetic divergence observed within these bacteriophages.

Synteny analysis and genome rearrangements in A. baumannii phages

To investigate the genomic architecture and evolutionary conservation of Acinetobacter baumannii phage φAB440, we conducted a comparative synteny analysis against twenty related Acinetobacter phages using BLASTp alignments. The panels (a–t) in Fig. 7 represent a Circos map depicting pairwise comparisons of φAB440 (blue semi-circle in each plot) with a distinct comparator phage genome (green semi-circles). The red connecting chords represent homologous proteins, indicating conserved syntenic regions based on amino acid sequence similarity. Highly interconnected plots with dense, straight chords in A-E (vB_AbaP_IME546, vB_AbaP_ZHSHW, vB_AbaP_APK48-3, vB_AbaP_B09_Aci08, vB_AbaP_APK14) reflect a high degree of synteny. Panels h (vB_Ab_164_KEN_01) and j (vB_Ab_164_KEN_02) show only a small number of homologous regions with limited coverage and fragmented alignments. Panel n (vB_Ab_1137_KEN_02) with cross-linking chords represents structural discontinuities. In Panel m (vB_AbaP_D2M) and Panel s (AB_NC12), the chords are sparse and less aligned, indicating partial homology with considerable divergence.

Fig. 7 — Circos maps depicting the BLASTp alignment results with respect to *A. baumannii* phage φAB440. In each panel blue-colored semi-circular segment represents φAB440 and green-colored segments represent different *Acinetobacter* phage genomes. a vB_AbaP_IME546. b vB_AbaP_ZHSHW. c vB_AbaP_APK48-3. d vB_AbaP_B09_Aci08. e vB_AbaP_APK14. f vB_Ab_1137_KEN_05. g vB_AbaP_APK48. h vB_Ab_164_KEN_01. i vB_Ab_164_KEN_03. j vB_Ab_164_KEN_02. k vB_AbaP_APK116. l Ab124. m vB_AbaP_D2M. n vB_Ab_1137_KEN_02. o Abp1. p phiAB6. q phiAB1. r SH-Ab_15519. s AB_NC12. t SWH-AB-1. The numbers on the external surface of segments indicate genome size in kb. The red colored chords in each panel indicate sequence conservation and orientation of those sequences with respect to φAB440 genome.

The synteny results for phage φAB441 and nine other related Acinetobacter phages are visualized using Circos plots in Fig. 8, where the reference genome φAB441 is represented in blue, and the compared genomes are represented in green. Panels a–c (vB_AbaM_B9, BS46, TaPaz) exhibit extensive collinearity with φAB441, as evidenced by numerous straight, criss-cross red chords extending across both genomes, indicating a high degree of genome-wide identity and conserved gene order, suggesting close evolutionary relationships. Panel d (Phab24) is distinguished by its extensive genomic rearrangements, indicated by numerous parallel chords between the genomes suggesting multiple inversions, translocations, or mosaic genome structures. Panels e–i (AM24, YMC13, P577, Herod, and Bestia) demonstrate reduced yet structured synteny with φAB441, with fewer and more scattered modules of homologous regions interspersed with large non-homologous sequences.

Proteome co-localization across Acinetobacter baumannii phages

Co-localization of key phage proteins across phage φAB440 and a panel of genetically diverse phages, is represented using Circos alignment (Fig. 9a). Each colored chord represents a high-confidence amino acid homology between φAB440 and another phage, with the color denoting the predicted function of the protein (as per the accompanying legend). Core structural modules are highly conserved. In case of comparison with phage φAB440, major capsid proteins (yellow arcs) and TSPs (olive arcs) show broad conservation across multiple phages, including PP841132, NC041905, and PP841139. Lysis module conservation is shown to vary among these phages. Endolysins (cyan) and holins (lime), which form the canonical lysis cassette, are present in only a subset of the compared phages. Proteins of replication machinery show selective conservation among Acinetobacter phages. DNA polymerase genes (orange arcs) are conserved primarily in phages like NC041905 and PP841135, suggesting that only certain members share a replication strategy similar to φAB440. Terminase subunits, essential for DNA packaging, show a split pattern: Small subunit terminase (blue-green) and large subunit terminase (purple) homologs cluster around a few phages. Phages NC041905 (SH-Ab 15519) and PP841135 (vB_Ab_1137_KEN_04), and demonstrate the most complete homologous representation of φAB440’s (PP792915) core proteins, potentially marking them as close relatives or members of the same genus.

In case of φAB441 (Fig. 9b), the homologues of TSP were present in all 9 Acinetobacter phages used for comparison. These TSPs are interspersed in genomes not maintaining order conservation. In phage φAB441, TSP is located adjacent to endolysin, and so does in phages MZ043613 (TaPaz), MW316733 (Bestia), MH133207 (vB_AbaM_B9). The cell wall hydrolases are present in the genomes of five Acinetobacter phages other than φAB441. The genes for these proteins are present upstream of the terminase large subunit in all the six cases. Other important structural protein major capsid protein is located adjacent to the TerL. Phage φAB441 and related phages have multiple tail fiber proteins in their respective genomes that are mainly localized in near vicinity of TSPs, but there is no exact gene order conservation.

GC skew and codon usage

The overall GC content of the two phages varied, with φAB440 having 39.02% and φAB441 having 32.43% exhibiting distinct genomic compositions. Around 1 kb region near the start position of the genome of φAB440 was devoid of any coding sequences, thus being a potential candidate region for containing origin of replication. Based on the cumulative GC skew a putative origin of replication was predicted at ~750 bp, and a putative terminus was predicted at ~41,000 bp (Supplementary Fig. 7). Similarly, for φAB441 a putative origin of replication at ~11,850 bp and a putative terminus was predicted at ~76,300 bp (Supplementary Fig. 7). Neither of the two phages encoded any tRNA.

Both the phages showed a bias in codon usage (Fig. 10). Relative Synonymous Codon Usage (RSCU) analysis revealed significant deviations in codon preferences between the phages and reference A. baumannii strain. Respective codon usage and RSCU values of φAB440, φAB441 and AT19606 are presented in Supplementary Table 9. Phage φAB440 maintained bacteria-like GC content but avoided GC-rich codons [CTG (Leu), GCC (Ala), CGC (Arg)]. Phage φAB441 had a strong AT bias, with extreme preferences for rare codons. For Arginine φAB441 preferred AGA (∆RSCU + 1.65) but avoided of GC-rich codons CGC (∆RSCU −0.577 and CGG (−0.461). When observed for specific amino acids, φAB440 strongly preferred TTA for Leu (∆RSCU + 0.688) and avoided TTG (−0.511) and CTG (−0.287). Phage φAB441 has a milder bias but still avoided CTT/CTC for leucine (∆RSCU, −0.161/−0.063). Phage φAB440 also avoided CGC/CGG and preferred CGT/AGG for arginine. Both phages prefer GGT for glycine (∆RSCU + 0.822 in φAB440, +0.369 in φAB441) and avoided GGC (−0.611 in φAB440, −0.731 in φAB441). TAG is slightly preferred in phage φAB440 (+0.106) as a stop codon, whereas TGA is neutral in both.

Phage-mediated in vitro anti-bacterial and anti-biofilm assay

The phage φAB440 and φAB441 were tested against their MDR hosts to determine their lytic potential and anti-biofilm activity. Phage φAB440 restricted the growth of bacteria in planktonic cultures at MOIs 10¹ to 10⁻¹ even up to 24 h (Fig. 11a). Till 8 h, MOI 10⁻² and 10⁻³ performed well in keeping the check on bacterial population. After 24 h phage φAB440 at MOIs 10⁻², 10⁻³ and 10⁻⁴ showed 2, 1.8 and 0.9 log₁₀ decrease in bacterial count (Fig. 11c) as compared to the non-treated group, but were not able to completely stop the bacterial growth. Phage φAB441 completely suppressed the growth of bacterial cell till MOI 10⁻³ even up to 24 h of incubation (Fig. 11b). At MOI 10⁻⁴, however it couldn’t stop the bacterial growth but still led to significant reduction of 0.4 log₁₀ in CFU/ml (Fig. 11d).

Fig. 11 — a Time kill curve assay of phage φAB440 against VTCCBAA1521 and, b of φAB441 against Fo53-5BP at MOIs 10¹-10⁻⁴. c Viable residual cell count after 24 h of time kill assay of φAB440 and, d φAB441 at various MOIs 10¹-10⁻⁴. e Reduction in biofilm content as measured by residual viable cell count and CV assay after treatment with phage φAB440 and, f φAB441 of their respective multidrug-resistant *A. baumannii* hosts. The data on vertical bars with left Y-axis represents viable cell count and data on spherical sets with right Y-axis represent quantification of biofilm reduction by CV assay. Error bars depict standard error of mean. Significance levels are depicted as ****p < 0.0001.

Phage φAB440 when test for eradication of preformed biofilms, performed well by completely eradicating biofilms at 10⁹ PFU/ml and reducing viable cell count by 8.178, 8.362, and 5.782 log₁₀ values at 10⁸, 10⁷ and 10⁶ PFU/ml as compared to control group (Fig. 11e, left Y-axis). The quantification of biofilm mass showed more robust anti-biofilm activity of φAB440. Phage at concentrations of 10⁹, 10⁸, 10⁷, and 10⁶ showed 2.852, 2.842, 2.815, and 2.378 log₁₀ reductions in biofilm mass (Fig. 11e, right Y-axis). Phage φAB441 completely eradicated the 4-day-old biofilm at 10⁹ and 10⁸ PFU/ml. At 10⁷ and 10⁶PFU/ml it showed 8.31 and 6.17 log reduction (Fig. 11f, left Y-axis), respectively, of viable cell count in biofilms. φAB441 showed strong biofilm eradication ability by causing 3.469, 3.453, 3.446, and 3.13 log₁₀ reductions (Fig. 11f, right Y-axis) in biofilm quantity as calculated by measuring absorbance at OD_570nm after CV assay.

For a more empirical analysis of biofilm eradication ability of A. baumannii phages, scanning electron microscopy was performed for the MDR host Acinetobacter biofilms developed on urinary catheters with or without phage application. In case of VTBAA1521, biofilms after 4 days of maturation were developed on inner walls of urinary catheter (Fig. 12a, b) with cell-to-cell polysaccharide junctions clearly visible in multiple compact layers of biofilms. The burst and degraded cells of the biofilm matrix after 18 h of treatment with phage φAB440 is presented in Fig. 12c, d. Fo53-5BP, being a hypermucoviscous strain formed thick and persistent biofilms, which can be seen as heaps of biofilm matrices (Fig. 12e). Dense and intercellular junctions spanning the compact layers of biofilm are seen in Fig. 12f. After treatment with phage φAB441, a complete eradication of biofilm (Fig. 12g, h) is achieved mediated by their depolymerases activity. Absence of any viable cell after respective phage treatment in both the cases with VTCCBAA1521 and Fo53-5BP was confirmed by CFU assay further validating the excellent anti-biofilm activity of both the phages.

Fig. 12 — a Structure of biofilm formed revealing cell-to-cell exopolysaccharide junctions (11,000 X). b Mono layers of A. baumannii biofilm dwelling on surface of urinary catheter (6000 X). c Phage φAB440 mediated eradication of *A. baumannii* strain VTCCBA1521 biofilm from urinary catheter visualized by FE-SEM at 500 X, and d at 1000 X. Biofilm of *A. baumannii* strain Fo53-5BP on urinary catheter visualized by FE-SEM. e Densely packed multi layers of *A. baumannii* biofilm dwelling on surface (500X), f Structure of biofilm revealing cell-to-cell exopolysaccharide junctions (7000 X). g Phage φAB441 mediated eradication of *A. baumannii* strain Fo53-5BP biofilm from urinary catheter visualized by FE-SEM at 500 X, and h at 7000 X.

Therapeutic potential of A. baumannii phage in Caenorhabditis elegans

In the infection control group, all the nematodes died within 20 h (Fig. 13). In the group where nematodes were treated only with bacteriophage, the survival decreased gradually over days but was significantly longer than infection group. In 10¹⁰ PFU/ml concentration 10% nematodes survived till 28 h. In 10⁹ PFU/ml, 10% nematodes survived till 32 observable hours. In 10⁸ PFU/ml, 9% nematodes survived till 32 h; in 10⁷ PFU/ml, 4% nematodes survived. In 10⁶, 10⁵, 10⁴ PFU/ml, 16%, 15%, and 16% nematodes survived till 28 h. In treatment groups phage was able to increase survival of nematodes better when used at higher MOIs. In MOI 1000 and 100, significant increase in survival was observed during 8–32 h. In MOI 10 and 1, increased significant survival was observed during 8–28 and 8–24 h, respectively. In MOI 0.1 and 0.01 treatment groups, nematode survival increased significantly during 12–20 h. In MOI 0.001, significant increase in survival was observed during 12–16 h. Phage was able to increase survival of nematodes up to 75.97%, 73.97% and 73.7% and showed an 11.32, 11.05, and 11 fold lower mortalities at MOIs 1000, 100 and 10, respectively.

Discussion

The treatment of multidrug-resistant A. baumannii (MDR-AB) has become complicated owing to overuse or misuse of broad-spectrum antibiotics and rapid bacterial evolution¹, leading to prolonged hospital stays and increased mortality rates evoking alternate phage therapies¹⁷. Phages exhibit high specificity for their host bacteria due to the precise interaction between phage proteins and receptors on the bacterial cell surface. These cell surface receptors may include proteins, exopolysaccharide (EPS), lipopolysaccharides (LPS), capsular polysaccharides (CPS), teichoic acids, or other molecules characteristic of the bacterial cell wall or membrane⁴³. Phages equipped with depolymerase enzymes (in tail fibers/spike proteins) can overcome this defense by specifically recognizing and enzymatically degrading CPS, thereby gaining access to the bacterial cell surface to initiate infection⁴⁴. The degradation of CPS manifests itself in the form of a translucent zone around the plaque on bacterial lawn known as ‘halo zone’⁴⁵. In this study, both the phages displayed strong halo regions that kept on increasing even after several days of incubation at 37 °C (up to remarkable 50 days for φAB441) reflecting their exceptional biofilm-degrading capabilities. The structural diversity of CPS owing to differences in monosaccharides, linkages, and polymer lengths, necessitates phage depolymerases to be highly specific, restricting their host range. This is evident in φAB440 and φAB441, which, despite narrow host ranges, exhibit potent biofilm reduction, highlighting the uniqueness of their depolymerases.

The ability of a phage to form viable plaques at 80°C and pH2 is an extreme phenotype suggesting remarkable thermostability of phage structural proteins. Thermo-tolerant and acidophilic phages have previously been isolated from extreme environmental sources like geothermal areas, hot springs, hydrothermal vents, compost piles, greenhouse soils etc. but reports of such phages from community sewage are fewer in number^46,47. Phages φAB440 and φAB441 in this study have been isolated from community sewage. It has been reported that formation of disulfide cross linking, β-barrel, and metal ion coordination in domains of structural proteins of bacteriophages is responsible for tolerance or stability of phages against thermal or acidic inactivation^48–51. Characterization of thermostable phages is important due to their applications in phage display systems, phage-based nanomedicine, phage-based probes for diagnostics^52,53. Trimerization of the tail fibers is also linked with the higher stability³¹. These structural characteristics and evolutionary relationships of functional domain associated with depolymerases are still not well understood. In this study, we have utilized the tools like Deepbinner, Porechop, Nanofilt, and Commander for genome assembly, AlphaFold for predicting complex structural information, PrankWeb and DoGSiteScorer to predict binding pockets, MEGA for phylogenetic analysis, and Circos for synteny analysis. By integrating these predictions, we tried to gain a deeper understanding of the molecular basis of stability and specificity of phages particularly for receptor-binding- and lysis- proteins that facilitate phage attachment and lysis of host cells, which is crucial for broadening the applicability of phage therapy. The structures of depolymerases predicted by AlphaFold were modeled in stable trimeric forms indicating their stability under extreme conditions.

Depolymerases usually have two main forms: lyases and hydrolases²¹. Some recent studies have identified several functional domains associated with the depolymerase activity of Acinetobacter bacteriophage tail fibers predominantly being Pectin lyase-like domain (PL), phage_tailspike_middle domain (PTM), Transglycosidases domain (TG), and SGNH hydrolase domain (SH)^54,55. These domains are primarily found in phages from the Autographiviridae family, specifically within the Friunavirus genus⁵⁵. Phylogenetic classification of φAB440 placed it in Friunavirus genus (Fig. 6e) but the maximum ANI value of 93.38% indicated a different species level. φAB441 (max ANI 92.24%) was found similar to other unclassified Caudoviricetes require further analysis for its precise classification. The phylogeny classification with VICTOR using whole genome highlights distinct evolutionary trajectories among Acinetobacter phages, with φAB440 and φAB441 exhibiting divergent clustering despite shared geographic origins implying niche-specific selective pressures or host-range specialization. The monophyletic grouping of φAB440 along with phages of the KEN Cluster, which have been isolated from and used in clinical settings⁵⁶, suggests strong conserved functional or structural adaptations, possibly linked to host infection strategies. This makes φAB440 a strong candidate for designing phage-based therapeutics especially in biofilm-related infections. Despite sharing ecological niche with φAB440, φAB441was an outlier along with Enterobacteria phage HK620. The tight clustering of the Acinetobacter phage in two major clades implies host genus-specific evolution, corroborating studies showing phages often co-diversify with bacterial hosts⁵⁷. The high genetic diversity observed (28 species-level clusters for φAB440-related phages vs. 17 for φAB441) suggests differing evolutionary rates or recombination frequencies among Acinetobacter phages.

Apart from whole genome or proteome, MCPs are also widely used for phage phylogeny due to their universal presence in tailed phages (Caudoviricetes) and structural conservation^58,59. On the basis of MCP analysis, φAB440 was placed distantly from other compared phages indicating a unique divergence in evolution. However, φAB441 found to be most closely related to vB_AbaM_B9, followed by MD-2021a, which is in alignment with the results from proteome analysis.

Presence of heptad repeats in coiled coil region in TSP of φAB440 and abundance of alanine contribute to high stability of the phage action, a feature also reported in thermostable Salmonella phage P22⁶⁰.

CDS46 of φAB440, encodes a chitinase class I domain from GH19 family, hydrolyzing β-1,4-N-acetyl-D-glucosamine linkages⁶¹. Family 19 chitinases are very rare in bacteriophages. Unlike lysozymes, GH19 chitinases lack sequence homology but shared structural similarity with phage T4 lysozyme^34,62. Absence of CBD domain classify it as globular endolysin, common in phages against Gram-negative bacteria³⁵. Such endolysins (from phages T1, P1, λ) often rely on short SAR (Signal Anchor Release) motifs for receptor binding^63,64. Though no classical SAR motif was detected, an N-terminal hydrophobic patch suggests membrane association, similar to phage T7 endolysins. Endolysin of φAB441, has a dual aromatic motif (Trp74/Tyr75), where indole ring of Trp mediates van der Waals interactions with glycan strands of peptidoglycan, enhancing binding affinity²³ and Tyr’s hydroxyl group forms hydrogen bonding with oxygen atoms of peptidoglycan thus help in aligning the substrates for cleavage⁶⁵. Tryptophan and tyrosine have been reported to contribute to the hydrogen bond network and are specifically positioned to interact with the peptidoglycan⁶⁶. The presence of glutamate as a catalytic residue on the active sites in endolysin of both the phages, validate the prediction probability and binding potential of active sites. In silico molecular docking of various components of biofilm matrix and cell wall, respectively, with TSPs and endolysins further validated their potential for therapeutic use. TSPs of both the phages exhibited high-affinity binding to diverse polysaccharide structures (ΔG = −9.2 to −4.6 kcal/mol). TSPs also showed strong affinity towards to A. baumannii outer membrane proteins (CarO/1/2) and biofilm response regulators (BfmR) (ΔG −84.9 to −63.9 kcal/mol) indicating potential exposure of these membrane porins as secondary targets after EPS stripping. This bipartite affinity of phage TSPs underscores exceptionally strong biofilm-eradicating potential. These indications open a new research arena to explore relation between phage TSP and bacterial porins blockade. Endolysins showed strongest interactions with NAG-NAM tetramers (ΔG = −7.86 kcal/mol), followed by NAG, NAM, NAM-L-alanine. These interactions support TSP-endolysin cocktails for treating recalcitrant biofilms persistence in A. baumannii infections. This comprehensive elucidation of structural units of phage proteins can be utilized for directed mutagenesis of tail fiber binding loops, binding pockets, replacement of tail components, trading tail fibers by directed homologous recombination⁶⁷ to reprogram and engineer broad-acting peptides as novel anti-biofilm agents.

Synteny represented through Circos plots allows for visualization of gene collinearity (conserved gene order), inversions (crossed lines indicating reverse orientation), insertions/deletions (regions without corresponding homologs), and modular genome organization (conserved functional blocks). This analysis highlights the mosaic and modular nature of phage genomes, where conserved core genes involved in replication, morphogenesis, and host lysis are preserved across related phages, while variable regions often encode accessory genes, including those related to host range, DNA modification, or anti-defense systems. High collinearity of φAB440 with vB_AbaP_IME546, vB_AbaP_ZHSHW, vB_AbaP_APK48-3, vB_AbaP_B09_Aci08, vB_AbaP_APK14 phages, sharing not only gene content but also gene order and orientation suggest strong evolutionary conservation and possibly shared ecological or host-related niches. Lower coverage and divergence of φAB440 with phages from KEN cluster (Fig. 7h, j, n) and others (Fig. 7m, s) indicates low protein homology and possible divergence at the genus or family level due to inversions, differential acquisition or loss of accessory genes or adaptation to different A. baumannii strains. This genome modularity is common in phages undergoing rapid evolution. The synteny analysis underscores the genomic plasticity of φAB441 and its relatives. Phages with high collinearity (e.g., vB_AbaM_B9, BS46) may belong to the same species or genus, sharing conserved core functional modules, particularly in genes encoding structural proteins and replication machinery^68,69. In contrast, phages like Phab24 and AM24 reflect localized genomic rearrangements or inversion events indicative of divergent evolutionary trajectories and potentially distinct host specificities or infection mechanisms⁷⁰. Significant genomic divergence with rest of the studied phages is likely due to the gain/loss of genetic material and/or extensive reshuffling of gene blocks. This pattern supports a mosaic genome architecture, which is a hallmark of tailed bacteriophages and often results from horizontal gene transfer and recombination across distant phage lineages⁶⁸. These differences reflect adaptations to diverse ecological niches or divergent phage-host co-evolutionary pressures^71,72. The observed genomic rearrangements are consistent with the modular theory of phage evolution, where core genes are retained for essential functions, while accessory genes, often associated with host interaction or environmental adaptability, undergo frequent exchange^73,74.

In phage φAB440, broad conservation of major capsid proteins and TSPs across multiple phages reflects evolutionary constraints on structural components critical to virion assembly and host recognition, suggesting a shared infection strategy⁶⁸. The frequent presence of TSP homologs suggests conservation in host receptor binding domains, possibly indicating a shared or overlapping host range. Limited conservation of canonical lysis cassette implies either divergence in lytic strategies or differences in host cell wall composition necessitating functional diversification⁷⁵. Conservation of DNA polymerases in few phages and absence in others may indicate reliance on host-encoded enzymes or divergent replication modules. Clustering of terminase sub-units around a few phages, suggest modularity in packaging strategies, consistent with known diversity in phage terminase systems⁷⁶. The mosaicism in genome architecture of Acinetobacter phages seen here with proteins of the same class found in different subsets of phages validates the modular theory of phage evolution, wherein exchangeable functional blocks are conserved in patches depending on evolutionary or ecological pressures⁷³.

In case of φAB441 and similar phages the physical linkage of depolymerase and lysin genes in phage genomes confers a selective advantage by coordination of two important steps of phage infection i.e. biofilm penetration and host lysis⁷⁷. This physical linkage ensures co-expression of both the genes. One single operon allows synchronized production of both the enzymes mediated by shared regulatory elements thus maximizing the efficiency²⁷. Lysins and tail spike proteins have been reported to be conserved together in many phages infecting biofilm-forming bacteria [Pseudomonas phages PB1-gp109, 110; PaP1(gp76, 77); LUZ19 (gp44, 45)] suggesting a positive selection for the linkage⁷⁸. Moreover, engineered phages with fused depolymerase-lysin genes have shown show 2–3 times greater biofilm clearance than wild-types⁷⁹ further validating the selection bias. Localization of CWH upstream of the TerL serves their synchronized expression, preventing premature lysis before genome packaging⁴⁰. Also, the shared promoters reduce transcriptional delays and helps increase regulatory efficiency³⁷. Positioning of the MCP downstream of the TerL in phage genomes is a conserved genomic order feature with critical functional and evolutionary implications. This is well established that terminase is part of the genes that express early in phage life cycle and MCP in late expressed genes. This downstream localization of MCP under late promoter lead to its late expression after TerL, to prevent premature capsid assembly^80–82. The physical proximity of tail fiber and tail spike genes in phage φAB441 other phage genomes reflects their functional cooperation in host recognition and infection, while the lack of strict gene order conservation suggests evolutionary flexibility to adapt to diverse hosts. Proximity allows shared regulatory elements, such as same promoters to achieve synchronized expression²⁷. As both proteins assemble into the phage baseplate, so low genomic spacing may facilitate their proper folding and interaction.

The average GC content of A. baumannii species has been reported to be around ~39%⁸³. The GC content of the phages and their host genomes become more similar over extended co-evolution. The GC content convergence of φAB440 (39%) with average A. baumannii GC content indicates long-term adaptation of the phage. Whereas the difference in GC content of phage φAB441 (32.4%) indicates a strongly AT-biased genome which tends to exhibit faster rates of genomic evolution and hence indicate more recent host jump. A clear, V-shaped cumulative GC skew curve is associated with theta-type bidirectional replication, commonly observed in circular genomes of many phages and bacteria^84–86, thus indicating the circularity of the isolated phage genomes. The presence of tRNA in a phage genome has been correlated with higher replication efficiency and host diversity^87–91. Thus, the absence of tRNA in phages φAB440 and φAB441 could explain a comparatively longer latent periodsand their narrow host ranges.

The complete absence of phage-encoded tRNAs in genomically compact phages (common among Autographiviridae) necessitates precise codon-host tRNA co-adaptation. Phage φAB440’s preference for TTA-leucine over CTG-leucine may reflect biofilm-specific optimization, as nutrient limitation in biofilms can dramatically alter tRNA modification profiles and translational efficiency⁹². Meanwhile, phage φAB441’s extreme AT-bias (32.43% GC) and AGA-arginine specialization (+1.65 ∆RSCU) could represent adaptation with selective pressure to utilize biofilm-induced tRNA pool alterations⁹². Collectively, these findings suggest that phage φAB440 may be more translationally optimized for its A. baumannii host than phage φAB441, which shows signs of codon mismatch and possible broader or recent host adaptation, a trend that we have continuously observed in this study. Phage φAB440’s host-mimicking yet competitive codon usage suggests efficacy against planktonic cells, while phage φAB441’s rare codon specialization may enhance penetration of the biofilm extracellular matrix where tRNA availability differs substantially⁹². The stark AGA-arginine bias in phage φAB441 particularly warrants investigation as a potential biomarker for biofilm-adapted A. baumannii strains, possibly explaining its narrow host range. From an evolutionary perspective, these phages demonstrate how codon usage can serve as both an adaptation mechanism and host-range determinant in the absence of tRNA genes. The findings support the growing recognition of codon-mediated regulation in phage life cycles⁹³.

We also validated the structural features of phage depolymerase and endolysin domains with their action in vitro to kill bacterial cultures and eradicate biofilms formed on urinary catheters. Both phages φAB440 and φAB441 have strong lytic profiles in terms of reduction of viable bacterial counts. Phage φAB440 suppressed the bacterial growth at MOIs 10¹-10⁻¹ and φAB441 at 10¹-10⁻³. No re-growth of bacterial cells at these MOIs suggests that along with strong lytic action, these phages are able to counter the emergence of resistance evolution at even at lower MOIs. When these phages were applied to test their anti-biofilm activity, a complete eradication was achieved at 10⁹ PFU/ml (with φAB440) and 10⁹-10⁸ PFU/ml (with φAB441). Although lower phage concentrations didn’t completely eradicate biofilms, they significantly reduced viable cell counts (p < 0.0001). The FE-SEM visualization of biofilm eradication from urinary catheters also revealed a complete eradication within 18 h of treatment. Phage φAB441 extended C. elegans survival against A. baumannii by up to 76% at high MOIs (1000-10), with 11-fold mortality reduction. This is the first report of using A. baumannii bacteriophage as a therapeutic in C. elegans model. Against Pseudomonas aeruginosa infection in C. elegans, phages have previously demonstrated 90% increased survival at MOI 100 and 80% increased survival at MOI 10⁹⁴. A 25% mortality reduction was also observed with Burkholderia pseudomallei bacteriophage therapy in C. elegans⁹⁵. Transient protection at lower MOIs is observed likely due to phage amplification limits amid high bacterial loads. In the current study, high bacterial load in C. elegans is also evidenced by death of all nematodes in around 20 h. of infection in control group. However, protection at lower MOIs also suggests biofilm breakdown or immunomodulatory effects observed beyond cell lysis. Phages have been shown to suppress pro-inflammatory cytokines such as interleukin 6 (IL-6), IL-8 and tumor necrosis factor alpha (TNF-α), IL-1β, nuclear factor-κB (NF-κB), C-X-C motif chemokine ligand 12a (CXCL12a), Toll-like receptor 4 (TLR4) activity and the overall inhibition of T cell activation in both in vitro and in vivo models^96–101. Also, phages have been reported to upregulate anti-inflammatory factors like suppressor of cytokine signaling 3 (SOSC3), IL-1 receptor antagonist (IL1RN) and IL-10^100,102. These effects reduce inflammation at low doses of phages without needing initial high phage titre. Demonstrating phage therapy for A. baumannii infection in C. elegans predicts real-world success for resistant strains of bacterial pathogens.

Previously, we have also established the potential of using A. baumannii bacteriophage in conjugation with antibiotics to enhance the biofilm eradication of MDR strains¹⁰³. Though use of bacteriophages to eradicate biofilm is still in experimental or pre-clinical stages, but few phages, phage cocktails and phage-derived proteins have been demonstrated as potential therapeutic candidates. Depolymerase from phage φAB6 has been demonstrated to degrade preformed biofilms at a concentration ≥0.78 ng¹⁰⁴. Another novel phage-based strategy is the development of a cationic photosensitizer and bacteriophage-based photodynamic antimicrobial agent (APNB) for enhanced biofilm ablation against A. baumannii¹⁰⁵. The synergy provided by the dual action of traditional antimicrobials and phages help reduce the minimum inhibitory concentrations, re-sensitize the antibiotics, provide evolutionary tradeoffs^106–108. However, the basic requisite for developing a phage-based therapeutic is essentially the robustness of the bacteriophage isolate. The antibacterial action of the bacteriophages isolated in this study is comparable to that of the previously isolated phages. Phage φAB440 suppressed complete bacterial growth up to 24 h without any resistance development till MOI 10⁻¹ and φAB441 till MOI 10⁻³. Only at lower MOIs of 10⁻²–10⁻⁴ phage φAB440 led to development of resistance and at MOI 10⁻⁴ for φAB441. One study reported a phage P1068 isolated against A. baumannii was not able to suppress complete bacterial growth even at higher MOI of 10 against various bacterial hosts¹⁰⁹. In another study, phage vB_Aba_QH4 reduced the A. baumannii growth up to 90%, but after 6 h the bacterial growth saw slight increase even at MOI 100¹¹⁰. KARL-1 was able to suppress bacterial growth at MOI 10⁻⁶, but a slight increase in bacterial O.D. was observable at MOI 10¹¹¹¹. While phages against A. baumannii are being frequently isolated but phages with confirmed depolymerase activity due to presence of TSPs are uncommon. This depolymerase activity is manifested in form of strong halo region surround the bacteriophage plaque on a bacterial lawn⁴⁵. We studied plaque morphologies of various bacteriophages from data published in public domain and found that only very few phages formed halo region indicative of depolymerase activity^112–115. The sustained expansion of halo zone for such a prolonged period of 11 and 50 observable days is a unique hallmark that distinguishes phages φAB440 and φAB441 from previously reported phages in literature where halos are typically static or show a short-term growth. The stable phages isolated in this study φAB440 and φAB441 with, higher thermotolerance, low pH sensitivity not only make them valuable for industrial/biotechnological applications but their extraordinary biofilm degradation capabilities as observed by increase in plaque halo, decrease in viable cell counts in planktonic as well as in biofilm cultures, eradication as visualized by FE-SEM, and the structural validation of phage enzymes, are in synchronization with each other and prove potential of these A. baumannii phages as strong candidates for their use in therapy.

The findings from this study conclude that the phage depolymerases represent a promising key to dismantle biofilms from medical devices. The sequence, structure and enzymatic activity of phage proteins can be mimicked or harnessed to design novel anti-resistant anti-biofilm compounds. These short motifs or catalytic residue-containing regions can be tweaked as per suitability to increase host specificity, stabilize binding interactions or eradicating polysaccharides of diverse nature including EPS, CPS, and LPS. The predicted binding sites in depolymerases may explain strain-specific activity against A. baumannii capsular polysaccharides, while endolysin motifs could guide engineering of broad-spectrum antimicrobials. Unlike conventional antibiotics, which struggle against biofilm-mediated resistance, depolymerase- and endolysin-inspired therapeutics could enhance drug penetration and efficacy, offering a strategic solution for persistent biofilm infections.

Methods

Antibiotic resistance profiling of bacterial strains using disk diffusion method

Total 11 A. baumannii strains were procured from Bacteriophage and Bacterial repositories of National Center for Veterinary Type Cultures, ICAR- National Research Center on Equines, Hisar, Haryana, India and were tested for antibiotic susceptibility using disk diffusion method as per CLSI guidelines¹¹⁶. Description of bacterial strains is mentioned in Supplementary Table 1. Bacterial strains grown in Mueller Hinton Broth No. 2 Control cations (CAMHB) medium were streaked to prepare a bacterial lawn on 4 mm thick Mueller Hinton agar (MHA) plates. Antibiotic discs (HiMedia, USA) were fixed on the agar and incubated overnight at 37 °C. Bacterial strains were classified for their resistance profile after measuring the diameter of the clear zone of lysis^32,116,117. All strains were grown in nutrient broth (NB) medium and preserved at −80 °C as 50% glycerol stocks.

Assessment of biofilm-forming ability of bacterial strains

Biofilm formation ability of Acinetobacter strains was assessed by the tube method¹¹⁸ with few modifications. Early log phase cultures of bacterial strains were used to inoculate 2× LB + 1% glucose medium in glass tubes. The tubes were kept in a static incubator for 4 days at 37 °C. After incubation the planktonic cultures from the tubes were discarded off and adhered biofilm on inner walls of tubes were washed with PBS, fixed with methanol, and finally stained with 0.4% crystal violet. The thickness of stained film on the inside of tube walls was used to measure the strength of biofilm formation and bacterial strains were categorized as strong, moderate, weak or non-biofilm formers.

Bacteriophage isolation, propagation and preservation

Bacteriophages were isolated using the enrichment method described previously¹¹⁹. A. baumannii strains VTCCBAA1521 and Fo53-5bp (laboratory isolates) were used as host bacteria. Enrichment reaction was prepared by mixing centrifuged and filtered community sewage water (from Hisar city, India) with log phase bacterial cultures separately and incubated overnight with vigorous shaking at 37 °C. Resulting phage lysate was centrifuged and filtered through a 0.22 μm PVDF (Polyvinylidene difluoride) syringe filter and spotted on host bacterial lawn plated using the double-layer agar (DLA) method¹⁰³. The filtrate positive for bacterial lysis were used for phage isolation after five rounds of successive plaque purification in saline magnesium (SM) buffer [NaCl – 5.8 g/L; MgSO₄ – 2.0 g/L; 1 M Tris (pH 7.5) – 50 ml/L, gelatin (2%) – 5 ml/L] using double-layer agar (DLA) method. High volume phage lysates were prepared and treated with NaCl and PEG to obtain purified concentrated phage suspensions¹²⁰. Bacteriophages extracted in SM buffer were stored as glycerol stocks at −80 °C and were accessioned as φAB440 and φAB441 in the Bacteriophage Repository at NCVTC, Hisar, Haryana, India.

Assessment of physiochemical stability of bacteriophages

Bacteriophage suspensions were individually incubated at varying temperatures (4–80 °C) to assess thermal stability, and combined with pH buffers (pH 2–10) in a 1:10 (v/v) ratio to test pH stability. To test chloroform sensitivity, phage suspensions were exposed to chloroform at final concentrations of 1%, 5%, 10%, and 20% (v/v). Following incubation at room temperature for 1 h, each treated sample was serially diluted and plated via the DLA method to enumerate viable plaque-forming units (PFU/mL). All assays were performed in triplicates.

Growth kinetics of bacteriophages

Growth kinetics of phages φAB440 and φAB441 were determined using a modified protocol by Zhao et al.¹²¹. One ml of each phage suspension (10⁶ PFU/mL) was mixed with host bacterial culture at MOI 0.001 for 8 min at 4 °C. Un-adsorbed phages were removed by centrifugation and re-suspending them in fresh NB. Samples (200 μL) were collected at every 3 min interval for 60 min and immediately enumerated by DLA method assay. The latent period was determined from the initial plateau phase, while burst size was calculated as the ratio of final progeny phage titre to initial infected cell count. Triplicate experiments were performed for each phage.

Host range and cross infectivity of bacteriophages

The host range of phages φAB440 and φAB441 was evaluated against eleven Acinetobacter strains using spot assay. Log-phase bacterial cultures (300 µl) grown in nutrient broth were combined with molten soft agar (3 ml) and overlaid on nutrient agar plates. Ten-fold diluted phage suspensions in SM buffer (10 μL) were spotted onto the solidified bacterial lawns. Following overnight incubation at 37 °C, lytic activity of phages was assessed based on plaque clarity and categorized as: no lysis, mild, moderate, or strong lysis, according to Kutter¹²².

Structural morphology of bacteriophages by transmission electron microscopy

To study structural morphology of bacteriophages, transmission electron microscopy (TEM) was performed. For visualization, 10 μl of purified phage suspension (1 × 10¹⁰ PFU/ml) was loaded on a 400-mesh carbon-coated nickel tabbed grid (Beta Tech) and was negatively stained with 2% uranyl acetate (pH4) for 1 min. After removal of excess stain with filter paper tip, grid was placed inside and observed using a transmission electron microscope (JEOL JEM-1011, Jeol, USA) with an accelerating voltage of 80 kV.

Phage DNA extraction, library preparation and whole genome sequencing

Bacteriophage DNA was extracted using a previously described protocol¹¹⁹. The concentration and quality of extracted DNA was checked using Qubit 4 Fluorometer and nanodrop spectrophotometer. Whole genome sequencing of bacteriophage was carried out using Oxford Nanopore MinION (Oxford Nanopore Technologies, Oxford, UK) technology. Sequencing libraries for nanopore sequencing were end primed by using NEBnext ultra II end repair kit, and cleaned up with AMPure XP beads. Native barcoding of end primed sample was carried out with NBD-V24 (ONT). Finally, equimolar concentration of barcode sample was ligated with sequencing adapter and purified with AMPure XP beads and eluted in 15 µl of elution buffer. The barcoded samples in buffer were loaded into MinION Flow Cell R10 Version (FLO-MIN114) and run in MINion MK1B sequencer at 100X coverage. Raw reads obtained from sequencing were de-multiplexed using Deepbinner https://github.com/rrwick/Deepbinner)¹²³. Porechop (https://github.com/rrwick/Porechop) and Nanofilt (http://github.com/wdecoster/nanofilt)¹²⁴ were used for trimming the raw reads and to assess their quality. The long reads obtained from Nanopore sequencing were assembled by using Commander v2.0.20 (http://www.genotypic.co.in/commander/).

Genome annotation of bacteriophages

The assembled genomes of A. baumannii bacteriophages were analyzed by RASTtk¹²⁵ pipeline for predicting and annotating open reading frames (ORFs). ORFs were predicted and functionally validated by BLASTp against NCBI Clustered non-redundant (ClusteredNR) protein database with default parameters. Predicted protein sequences were searched on InterProScan¹²⁶ and CDD⁸⁶ for conserved domain identification. Putative t-RNAs were identified using tRNAscan-SE and ARAGORN with default parameters^127,128. ARGs genes were screened by using RGI-CARD¹²⁹. PhageScope was used to predict the lytic/lysogenic nature of phages¹³⁰. Rho-independent terminators were predicted using ARNold¹³¹. Proksee was used for genomic map visualization¹³². The complete genomes of A. baumannii phage φAB440 and φAB441 were submitted to GenBank with accessions PP792915.1 and PV420835.1, respectively.

Endolysins, tail spike proteins (TSPs) and their molecular docking

Endolysins and TSPs encoded by the both Acinetobacter baumannii bacteriophages were identified through BLASTp homology searches against the NCBI ClusteredNR protein database. Three-dimensional structures of these proteins were predicted with AlphaFold3¹³³ selecting the top-ranked model with mean pLDDT >70 for high-confidence regions. The predicted models were further validated using SWISS-MODEL Workspace, with QMEANDisCo scores¹³⁴ indicating overall model quality, and the UCLA-DOE SAVES server (v6.1), which provided detailed assessments including ERRAT scores for non-bonded atomic interactions¹³⁵, VERIFY3D scores for residue environment compatibility^136,137 and PROCHECK analysis for stereochemical quality and Ramachandran plot statistics¹³⁸. Structures were refined and visualized in PyMOL (v2)¹³⁹. Structural superimpositions and RMSD calculations were conducted in PyMOL to assess geometric similarity with reference structures. Active sites were computationally predicted using PrankWeb¹⁴⁰, DoGSiteScorer¹⁴¹ and active sites with highest scores were selected. The modeled protein structures of Lysin and TSPs for both organisms were further examined for their binding with commonly found membrane glycoproteins, glucose, or lipid compounds. Hence, compounds i) N-acetylglucosamine (Pubhem CID 24139), ii) Pentaglycine (PubChem CID 81537), iii) NAG-NAM dimer (PubChem CID 72210857), iv) N-acetylmuramic acid (PubChem CID 5462244), v) NAM-L-alanine (PubChem CID 10970945), vi) NAG-NAM tetramer (extracted from PDB ID 4BPA), vii) Peptidoglycan-associated lipoprotein from Acinetobacter baumannii (PDB ID 4G4V), viii) Peptidoglycan-associated lipoprotein from Acinetobacter baumannii (PDB ID 4G4W), and ix) Peptidoglycan-associated lipoprotein from Acinetobacter baumannii (PDB ID 4G4X) were selected to check the binding affinity of the endolysin proteins; and compounds i) Capsular polysaccharide type 6, Actinobacillus pleuropneumoniae (PubChem CID 148055), ii) Capsular polysaccharide type 7, Actinobacillus pleuropneumoniae (PubChem CID 195740), iii) Capsular polysaccharide, Klebsiella k15 (PubChem CID 3083199), iv) Capsular polysaccharide K49 (PubChem CID 195668), v) Capsular polysaccharide K98 (PubChem CID 197416), vi) Escherichia coli serotype O86 O-antigen (PubChem CID 91862843), vii) OMPA outer membrane proteins (PubChem SID 135340124), viii) Acinetobacter baumannii outer membrane protein CarO (PDB ID 4FUV), ix) Acinetobacter baumannii CarO1 (PDB ID 4RL9), x) Acinetobacter baumannii CarO2 (PDB ID 4RLB), xi) N-terminal domain of BfmR from Acinetobacter baumannii (PDB ID 5HM6), xii) Acinetobacter baumannii outer membrane protein Omp33 (PDB ID 6GIE) were chosen for docking studies against TSP, against both organisms. For protein-protein interaction studies, the HDOCK¹⁴² server was utilized, while for protein-ligand interactions, AutoDock v4.2.6¹⁴³ was employed. For docking studies, proteins and ligands were energy minimized using UCSF chimera v1.7¹⁴⁴. Top-ranked dock conformations were picked for each dock complex. Ligplot+¹⁴⁵ was used for protein-ligand interaction analysis.

Phylogenetic and synteny analysis

To assess evolutionary relationships, phylogenetic trees of phage φAB440, φAB441, and other known A. baumannii phages were constructed using the Genome-BLAST Distance Phylogeny (GBDP) method in the VICTOR web tool¹⁴⁶. Intergenomic distances were calculated with 100 pseudo-bootstrap replicates, and a balanced minimum evolution tree was inferred with branch support via FASTME including SPR postprocessing (for the formulas D0/D4/D6). Taxon boundaries at the species, genus and family level were estimated with the OPTSIL program¹⁴⁷. Trees were rooted at the midpoint¹⁴⁸ and visualized with ggtree¹⁴⁹. Additionally, protein-based phylogenies were reconstructed using conserved phage genes: the major capsid protein (MCP) and terminase large subunit (TerL). Amino acid sequences were aligned using ClustalW¹⁵⁰ in MEGA12¹⁵¹, and phylogenetic trees were generated via the neighbor-joining method with 1000 bootstrap replicates for branch support. For synteny analysis of A. baumannii phages, Circos plots were generated with Circos v0.69-8¹⁵². Gene annotations (CDS) were extracted from genome submission files submitted in NCBI. BLASTp alignments were used to identify homologous regions. The circos configuration files were generated using PerL 5.038002, with karyotype files defining the genome sizes and ideograms. Outputs were cross-verified with Proksee genome browser¹³² to ensure accuracy in feature positioning.

GC skew and codon usage

GC skew and cumulative GC skew plots were generated using a custom Python script with a sliding window of 500 bp and a step size of 50 bp. Codon usage of the bacteriophages φAB440 and φAB441 was analyzed with Cusp from EMBOSS¹⁵³ by comparing them with that of Acinetobacter baumannii ATCC19606 (type strain).

In vitro antimicrobial assay of A. baumannii phages in planktonic cultures

Lytic efficiency of bacteriophages was tested using time kill curve analysis in planktonic cultures¹⁵⁴. Both the phages φAB440 and φAB441 were tested individually to determine the bacterial killing at different ratios. The output of lytic efficiency was calculated in terms of optimal multiplicity of infection (MOI) at which the phage titres in the resulting suspensions were maximum and the antibacterial efficiency in terms of reduced bacterial load after infection. Host bacterial cultures (100 μL, ~1 × 10⁷ CFU/ml) in CAMHB, HiMedia, USA, were mixed with respective phage suspension (100 μL) in SM buffer at MOIs 10¹-10⁻⁴ in the wells of a flat bottom 96 well micro test plate. Bacterial culture with sterile SM buffer in equal concentrations served as positive control and sterile CAMHB with sterile SM buffer were taken as negative control. Plates were incubated at 37 °C under shaking conditions in a Multiskan GO Microplate Spectrophotometer to determine time kill curves by measuring OD_600nm at every 30 min for 24 h using SkanIt™ Software (Thermo Scientific, ver. 1.01.12).

Phage-mediated eradication of A. baumannii biofilms from urinary catheters

The anti-biofilm efficacies of both the bacteriophages were tested quantitatively by crystal violet (CV) assay in 96 well U-bottom micro test plate and for visual confirmation field emission-scanning electron microscopy (FE-SEM) analysis were performed. Briefly, overnight-grown bacterial cultures of both the bacterial hosts, VTCCBAA1521 and Fo53-5BP in Luria Bertani (LB) broth were re-inoculated and incubated to achieve 0.5 McFarland standard equivalent growth. 200 µL from these cultures were separately added into the wells of the U-bottom plate and the plates were kept at 37 °C under static conditions to allow the biofilms to establish and media was replenished after every 12 h. After 4 days, the planktonic content was removed, and plate was washed 2× with sterile PBS. To the empty wells, respective bacteriophage suspensions were added in varying concentrations (10⁶, 10⁷, 10⁸, 10⁹ PFU/ml). After incubating for 18 h at 37 °C, the biofilm was stained with 0.4% CV, as described for tube method. The stain was then dissolved using 33% acetic acid and the contents from each well were transferred to a flat-bottom micro test plate to quantify the biofilm contents. For this, OD₅₇₀ nm was measured in a Multiskan GO Microplate Spectrophotometer, using SkanIt™ Software (Thermo Scientific, ver. 1.01.12).

For FE-SEM visualization of anti-biofilm action of phages, biofilms of MDR A. baumannii hosts were developed on the inner walls of silicone urinary catheters. Log phase culture of host bacteria LB broth were used to re-inoculate the wells of a 6-well culture plate with sections of urinary catheters (1 × 0.5 cm²) in wells with 2 × LB + 1% glucose and biofilms were allowed to develop for 4 days at 37 °C with media replenishment after every 12 h. Thereafter, the catheter sections were removed carefully and immersed in bacteriophage suspensions (5 ml, 10⁹ PFU/ml) for 18 h. Treatment with sterile SM buffer served as control. Cells adhered on the catheter sections were carefully scraped off and enumerated using colony-forming unit (CFU) assay. Another replicate set of catheter sections proceeded for biofilm visualization using FE-SEM. After washing with sterile PBS, sections were fixed in 2.5% glutaraldehyde for 1 h at 4 °C and dehydrated with ethanol gradient [25%, 50%, 75%, 90%, 100% (2X)]. Dehydrated catheter sections were fixed on a copper stub, sputter coated with gold for 1 min, and visualized at an accelerating voltage of 5–10 kV in a JSM-7610F Plus Scanning electron microscope, Jeol, Japan.

In vivo assay of A. baumannii phage in Caenorhabditis elegans

Caenorhabditis elegans nematode model was used to assess the potential in vivo efficacy of phage φAB441 against A. baumannii. Wild-type C. elegans (N2) synchronized at L4 stages were used in this study. Nematodes were maintained and propagated on nematode growth media (NGM) plates carrying E. coli OP50 as food source at 20 °C. Experiment was conducted in liquid-based assay. For this, nematodes were extracted from NGM plates and washed thrice with M9 buffer and added in the wells of 96 well microtest plate already containing host A. baumannii at 1 × 10⁷ CFU/ml suspended in Nutrient Broth and/or phages at various MOIs ranging from 1000 to 0.01 suspended in PBS. Each well received 20 worms suspended in 10 µl M9 buffer. The total volume in each well was kept at 150 µl. Nutrient Broth, PBS and M9 buffer collectively were used as negative control. The plates were incubated at 20 °C and survival of the nematodes was monitored every 4 h for 32 h. The results were evaluated based on live nematodes (moving or curved shaped) and dead nematodes (lack of movement or straight) count. The experiment was conducted in triplicates.

Statistical analysis

Statistical tests were performed with two-way factorial ANOVA using Tukey’s multiple comparisons test in GraphPad Prism v.8.0.2 (263) to validate significance of bacterial killing and biofilm eradication at varying phage titres. P-value < 0.01 was set as threshold for the significance.

Supplementary information

Supplementary information^{(1.7MB, docx)}

Acknowledgements

This study was funded by National Fellow Grant, Indian Council of Agricultural Research, New-Delhi, India, and Maharishi Kanad Post Doctoral Fellowship, Institution of Eminence, University of Delhi, India. The funders played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript.

Author contributions

M.V.: Conceptualization, Methodology, Software, Formal analysis, Investigation, Resources, Data Curation, Writing – Original Draft, Visualization. P.K.(c): Software, Validation, Visualization, Data Curation. P.K.(a): Software, Validation, Writing - Review & Editing. I.R.: Software, Writing - Review & Editing. P.S.: Data Curation, Writing - Review & Editing. K.B.: Data Curation, Writing - Review & Editing. D.B.: Investigation, Data curation. N.V.: Writing – Review & Editing, Validation, Supervision. B.C.B.: Validation, Writing – Review & Editing. R.K.V.: Validation, Writing – Review & Editing. M.S.: Methodology, Resources, Writing - Review & Editing. T.A.: Conceptualization, Validation, Formal analysis, Resources, Writing – Review & Editing, Supervision, Project administration, Funding acquisition.

Data availability

The bacteriophages isolated in this study were accessioned at Bacteriophage Repository at National Centre for Veterinary Type Cultures, ICAR- National Research Centre on Equines. The phage genomes are available in the NCBI GenBank with the accessions PP792915.1 (φAB440) and PV420835.1 (φAB441). The datasets (microbiome data) generated and analyzed during this study are included in this article and its supplementary information.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

The online version contains supplementary material available at https://doi.org/10.1038/s41522-026-01137-x.

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Eradication of catheter-associated MDR Acinetobacter baumannii biofilms mediated by novel phage encoded depolymerase activity

Medhavi Vashisth

Pawan Kumar

Prexha Kapoor

Indu Rani

Priya Sharma

Karan Bhutani

Dheeraj Bansal

Nitin Virmani

Bidhan Chand Bera

Rajesh Kumar Vaid

Meenakshi Sharma

Taruna Anand

Abstract

Introduction

Results

Characterization of Acinetobacter strains

Characterization of A. baumannii phages

Fig. 1. Plaque morphology of A. baumannii bacteriophages.

Fig. 2.

Genome analysis of A. baumannii phages

Fig. 3. Genome maps of Acinetobacter baumannii bacteriophages.

Analysis of key phage proteins

Depolymerase genes

Fig. 4. Modeled tertiary structures of lytic proteins of A. baumannii phages generated by AlphaFold3 and visualized using PyMOL.

Genes for host lysis/endolysins

In silico interaction of phage TSPs and endolsyins with bacterial receptors

Table 1.

Table 2.

Fig. 5. Ligand binding interactions of TSPs and endolysins of Acinetobacter baumannii bacteriophages.

Genes for DNA packaging

Fig. 6. Phylogenetic trees of A. baumannii phages.

Major structural genes

Evolutionary relatedness of phages

Synteny analysis and genome rearrangements in A. baumannii phages

Fig. 7. Synteny analysis of phage φAB440 with similar Acinetobacter phages.

Fig. 8. Synteny analysis of phage φAB441 with other Acinetobacter phages.

Proteome co-localization across Acinetobacter baumannii phages

Fig. 9. Genomic mapping and co-localization of functional proteins in Acinetobacter baumannii bacteriophages.

GC skew and codon usage

Fig. 10. Codon usage by Acinetobacter baumannii phages φAB440 and φAB441 phages as compared to the codon usage of Acinetobacter baumannii ATCC19606.

Phage-mediated in vitro anti-bacterial and anti-biofilm assay

Fig. 11. Antibacterial and anti-biofilm activity of A. baumannii bacteriophages.

Fig. 12. Biofilm of A. baumannii strain VTCCBAA1521 on urinary catheter visualized by FE-SEM.

Therapeutic potential of A. baumannii phage in Caenorhabditis elegans

Fig. 13. Therapeutic efficiency of Acinetobacter baumannii phage φAB441 against MDR A. baumannii Fo53-5bp infection in Caenorhabditis elegans.

Discussion

Methods

Antibiotic resistance profiling of bacterial strains using disk diffusion method

Assessment of biofilm-forming ability of bacterial strains

Bacteriophage isolation, propagation and preservation

Assessment of physiochemical stability of bacteriophages

Growth kinetics of bacteriophages

Host range and cross infectivity of bacteriophages

Structural morphology of bacteriophages by transmission electron microscopy

Phage DNA extraction, library preparation and whole genome sequencing

Genome annotation of bacteriophages

Endolysins, tail spike proteins (TSPs) and their molecular docking

Phylogenetic and synteny analysis

GC skew and codon usage

In vitro antimicrobial assay of A. baumannii phages in planktonic cultures

Phage-mediated eradication of A. baumannii biofilms from urinary catheters

In vivo assay of A. baumannii phage in Caenorhabditis elegans

Statistical analysis

Supplementary information

Acknowledgements

Author contributions

Data availability

Declarations

Competing interests

Footnotes

Supplementary information

References

Associated Data

Supplementary Materials

Data Availability Statement

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