Analysis of a new phage, KZag1, infecting biofilm of Klebsiella pneumoniae: genome sequence and characterization.

Among the 95 bacterial isolates obtained from sputum samples, 15 isolates (15.7%) displayed characteristic Klebsiella morphology, including mucoid appearance, lactose fermentation, and non-hemolytic on blood agar plates. These isolates were further selected for antibiotic susceptibility testing. The susceptibility of the isolated K. pneumoniae strains to various antibiotics was evaluated using the disc diffusion method. The results, shown in (Table  1 ), indicated that the K. pneumoniae strains exhibited resistance to more than two of the tested antibiotics. Notably, the strains showed resistance to multiple antibiotics, including gentamicin, sulfamethoxazole, tetracycline, amoxicillin/clavulanate, nalidixic acid, and cefotaxime. Among the tested isolates, K. pneumoniae K9, isolated from a sputum sample, displayed the highest Multiple Antibiotic Resistance (MAR) index, with a value of 1.0, indicating significant resistance to multiple antibiotics. Molecular identification of this isolate (K9) was performed by extracting genomic DNA, amplifying rRNA using PCR, and sequencing the amplicon. The obtained sequence was deposited in the GenBank under accession number OP942216 . Table 1 Antibiotic Susceptibility using disc diffusion method for Klebsiella pneumoniae Antibiotic Class Antibiotic Bacterial Isolates K9 5D 5A 5B 5C K8 6A 6B 6C 6D K2 K11 K12 K17 K20 Cephalosporines 2nd generation Cefoxitin (FOX 30µg) R R R R R R R R R R R R R R R Cephalosporines 3rd generation Cefepime (FEP 30µg) Ceftriaxone (CRO 30µg) Ceftazidime ( CAZ 30µg) R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R Aminoglycosides Gentamicin (CN 10µg) Amikacin (AK 30µg) R R R R S R S R R R S R R R I R R R R R I S S S S I S S R I Carbapenem Imipenem (IPM 10µg) R R R R R R S R I R S S S S S Flouroquinolones Ciprofloxacin (CIP5µg) R R R R R R R R R R S R R S R Macrolides Erythromycin (E 15µg) R R R R R R R R R R R R R R R Chloramphenicols Chloramphenicol (C30mcg) R R R R S R R S R R S R S S S Penicillins Piperacillin (PRL 100 µg) R R R R R R R R R R R R R R R Tetracyclines Tetracycline (TE 30mcg) R R R R R R S S S S S S S S R Sulfonamide Trimethoprime/ Sulphamethoxazole (SXT25 µg) R R R R R R R R R R R I S S R Lipopeptides Polymixin B (PB 300 IU) R R S S S S S S S R R R R R R Resistance percentage for each isolate (%) 100 100 85.7 85.7 85.7 85.7 78.5 71.4 78.5 92 57 64 57 50 78.5 MAR Index 1 1 0.8 0.8 0.8 0.8 0.7 0.7 0.7 0.9 0.5 0.6 0.5 0.5 0.7 Antibiotic Susceptibility using disc diffusion method for Klebsiella pneumoniae R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R S R S R R R S R R R I R R R R R I S S S S I S S R I Chloramphenicol (C30mcg) Trimethoprime/ Sulphamethoxazole (SXT25 µg) Bacteriophages specific to K. pneumoniae K9 were isolated from an enrichment culture containing K. pneumoniae . Sewage water samples were collected from two large sewage plants in Sharkia Governorate, following the procedures outlined in the materials and methods section. The spot and plaque assay methods were utilized to detect the presence of phages. Among the observed plaques, a single plaque was selected for further analysis. This plaque was designated as Zag1, and subsequent purification and characterization steps were conducted. Transmission electron microscopy (TEM) was employed to examine the morphology of the isolated phage, KZag1. KZag1 exhibits a typical Myovirus structure; featuring a 75 ± 5 nm diameter icosahedral head and a short tail measuring 15 ± 5 nm.The results demonstrated that KZag1 belongs to the Myoviridae family. (Fig.  1 ). Fig. 1 Morphological Characteristics of K. pneumoniae Phage KZag1 ( A ) Phage Zag1 Plaque Morphology NA double-layer agar plate showing the plaque morphology of phage Zag1. Clear zones indicate areas where phage KZag1 has lysed the host K. pneumoniae cells. B Transmission Electron Microscopy (TEM) Image of Phage Zag1 shows three individual phage Zag1 particles, with a scale bar of 0.5 µm. C Transmission Electron Microscopy (TEM) Image of Phage Zag1 High-resolution TEM image revealing the detailed structure of phage Zag1. Scale represents 100 nm. D Phage Zag1 Adsorption on K. pneumoniae Cell Visual depiction of phage Zag1 attached to the surface of a K. pneumoniae bacterial cell Morphological Characteristics of K. pneumoniae Phage KZag1 ( A ) Phage Zag1 Plaque Morphology NA double-layer agar plate showing the plaque morphology of phage Zag1. Clear zones indicate areas where phage KZag1 has lysed the host K. pneumoniae cells. B Transmission Electron Microscopy (TEM) Image of Phage Zag1 shows three individual phage Zag1 particles, with a scale bar of 0.5 µm. C Transmission Electron Microscopy (TEM) Image of Phage Zag1 High-resolution TEM image revealing the detailed structure of phage Zag1. Scale represents 100 nm. D Phage Zag1 Adsorption on K. pneumoniae Cell Visual depiction of phage Zag1 attached to the surface of a K. pneumoniae bacterial cell The infectivity of Zag1 phage was assessed against different strains of K. pneumoniae and other bacterial strains (Table  2 ). KZag1 phage displayed robust lytic activity specifically targeting K. pneumoniae (Table  2 ) . However, the remaining tested bacterial strains showed resistance to infection by the isolated phage, underscoring the highly specific nature of KZag1 towards K. pneumoniae . Table 2 Host Range of K. pneumoniae KZag1 phage Bacterial host Strain Spot test Klebsiella pneumoniae K9 (main host)  + ve K. pneumoniae K2  + ve K. pneumoniae K8 -ve K. pneumoniae K11 -ve K. pneumoniae K12  + ve K. pneumoniae K17  + ve K. pneumoniae K20 -ve K. pneumoniae 5A  + ve K. pneumoniae 5B  + ve K. pneumoniae 5C  + ve K. pneumoniae 5D  + ve K. pneumoniae 6A  + ve K. pneumoniae 6B  + ve K. pneumoniae 6C  + ve K. pneumoniae 6D  + ve Staphylococcus aureus saEg01LC596095 -ve Escherichia coli M30LC649234.1 -ve Escherichia coli ATCC25922 -ve Salmonella typhi ATCC14028 -ve Pseudomonas aeruginosa ATCC9027 -ve Host Range of K. pneumoniae KZag1 phage The one-step growth curve analysis of KZag1 phage revealed a characteristic pattern of phage infection and replication. The latent period, which represents the time between phage adsorption and the initiation of replication, was determined to be 20 min. This was followed by a rise period lasting 35 min, during which the phage population experienced exponential growth (Fig.  2 ). The entire cycle of infection, from adsorption to the release of new phage particles, was completed in approximately 50 min. The burst size, indicating the number of phage particles released per infected host cell, was calculated to be 83 for KZag1 phage. These findings provide valuable information about the kinetics and efficiency of phage replication and release within the host bacterial population. Fig. 2 Single-step growth curve for K. pneumoniae KZag1 phage. The plaque forming units (PFUs) per infected cell in cultures of K. pneumoniae K9 at different time post infection are shown. Samples were taken at intervals every 10 min Single-step growth curve for K. pneumoniae KZag1 phage. The plaque forming units (PFUs) per infected cell in cultures of K. pneumoniae K9 at different time post infection are shown. Samples were taken at intervals every 10 min The stability of Zag1 phage was assessed under different temperature and pH conditions. The results indicated that the infectivity of KZag1phage remained largely unaffected by temperature, particularly up to 60 °C (Fig.  3 A). The phage exhibited a survival rate ranging from 50 to 80% after exposure to temperatures of 70 °C for 10 min, suggesting its thermostability. However, at higher temperatures, KZag1 phage lost its infectivity and ability to lyse K. pneumoniae K9. Regarding pH stability, KZag1 phage demonstrated relatively stable behavior within the pH range of 6 to 8 (Fig.  3 B). At pH levels of 11 or higher and pH levels of 4 or lower, Zag1 phage completely lost its infectivity. Notably, the phage exhibited greater stability and infectivity at pH 7. These findings highlight the resistance of KZag1 phage to temperature variations within a certain range and its sensitivity to extreme pH conditions. Understanding the stability of KZag1 phage under different environmental conditions is crucial for its potential applications and efficacy in controlling K. pneumoniae infections. Fig. 3 Effect of temperature and pH on the stability of K. pneumoniae KZag1 phage. A The stability of phage of K. pneumoniae Kzag1 at different temperatures. B The stability of KZag1 phage at different pH values. The number of phage was estimated by plaque assay using K. pneumoniae . Results are shown as means ± standard error Effect of temperature and pH on the stability of K. pneumoniae KZag1 phage. A The stability of phage of K. pneumoniae Kzag1 at different temperatures. B The stability of KZag1 phage at different pH values. The number of phage was estimated by plaque assay using K. pneumoniae . Results are shown as means ± standard error In vitro experiments were conducted to assess the lytic activity of KZag1 phage against a highly multidrug-resistant strain of K. pneumoniae . When K. pneumoniae K9 was used as the host in combination with the isolated Zag1 phage, remarkably different inhibition patterns were observed. After a 24-h incubation period, complete inhibition of bacterial growth was observed, demonstrating the potent inhibitory activity of KZag1 phage against K. pneumoniae . The activity of Zag1 phage against K. pneumoniae K9 biofilm formation was investigated using varying MOI values (0.1, 1, and 10). The results showed a significant decrease in the biofilm biomass when treated with Zag1 phage compared to the control group (Fig.  4 ) among the tested MOI values; an MOI of 10 exhibited the highest inhibition of K. pneumoniae K9biofilm formation by the phage (Fig.  4 ). These findings indicate the strong potential of Zag1 phage in targeting and reducing biofilm formation by K. pneumoniae K9. This suggests its potential as an effective biocontrol agent to combat K. pneumoniae infections. Further studies are warranted to explore its applicability and efficacy in real-world settings. Fig. 4 Phage Treatment of K. pneumoniae K9 Biofilm. The figure demonstrates the impact of Kzag1 phage treatment on K. pneumoniae bacterial biofilms using different multiplicities of infection (MOIs) of 0.1, 1 and10. Each data point on the graph represents the mean of three independent experiments. The results indicate the efficacy of phage treatment in reducing K. pneumoniae biofilm formation at varying MOI values Phage Treatment of K. pneumoniae K9 Biofilm. The figure demonstrates the impact of Kzag1 phage treatment on K. pneumoniae bacterial biofilms using different multiplicities of infection (MOIs) of 0.1, 1 and10. Each data point on the graph represents the mean of three independent experiments. The results indicate the efficacy of phage treatment in reducing K. pneumoniae biofilm formation at varying MOI values The genome of the KZag1 phage, belonging to the Myoviridae family, was analyzed in this study. The phage genome had a size of approximately 157 kilobase pairs (kbp) and was found to contain 202 predicted ORFs (Table  3 ). Comparative analysis revealed a high degree of similarity between the KZag1 phage and Klebsiella virus 0507KN21.The genomic analysis of KZag1 revealed several notable features (Fig.  5 ). The total length of the genome was determined to be approximately 157 kbp, which is relatively large compared to other characterized phages. Within this genome, 202 ORFs were identified and annotated. These ORFs encode proteins with various putative functions, including those involved in phage replication, DNA packaging, structural proteins, and host interaction. A comparative analysis of the KZag1 genome with Klebsiella virus 0507KN21 revealed significant similarity, as depicted in (Fig.  6 ). These two phages exhibited a high degree of nucleotide sequence identity and displayed conserved genomic organization, suggesting a close evolutionary relationship and shared ancestry. Phylogenetic analysis further elucidated this connection by revealing a close relationship between KZag1 and other phages: Klebsiella phage T751, and Klebsiella phage cp18 (Fig.  6 ). The scale bar of 0.01 indicates minimal evolutionary divergence among these phages, implying a recent common ancestor. This observation is reinforced by the fast minimum evolution method employed in the pairwise alignments, which underscores the genetic similarity and shared evolutionary history among these phages. The high number of ORFs in the KZag1 genome indicates a complex genetic composition. These ORFs likely play crucial roles in the phage's lifecycle, including host recognition, replication, and assembly. The presence of specific genes associated with DNA modification, recombination, and mobile genetic elements suggests the potential for genetic diversity and adaptation within the phage population. Furthermore, the similarity to Klebsiella virus 0507KN21 suggests a shared host range and similar strategies for infecting and propagating within K. pneumoniae . This finding is significant as it indicates that KZag1 may possess similar infectivity and therapeutic potential against K. pneumoniae biofilms. The genomic characterization of KZag1 provides valuable insights into its genetic makeup and potential functions. The presence of numerous ORFs, similarity to Klebsiella virus 0507KN21, and the identification of specific genes involved in phage-host interactions highlight the phage's ability to infect and replicate within K. pneumoniae . Further investigation into the specific functions and mechanisms of these genes will deepen our understanding of phage-host dynamics and facilitate the development of phage-based therapies against K. pneumoniae infections. Table 3 Annotation table CDS Position of KZag1 ORF CDS Position BLAST Hit E-Value 1 complement(120..887) PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: ribonuclease; PP_00001; phage(gi100193) 0.0 2 929..1468 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00002; phage(gi100194) 2.50e-129 3 1472..2242 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: thymidylate synthase; PP_00003; phage(gi100195) 0.0 4 2229..3347 PHAGE_Serrat_vB_Sru_IME250_NC_042047: baseplate wedge; PP_00004; phage(gi100174) 0.0 5 3350..5677 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: dihydrofolate reductase; PP_00005; phage(gi100197) 0.0 6 5680..5835 hypothetical; PP_00006 0.0 7 5832..6152 PHAGE_Klebsi_2146_NC_049472: hypothetical protein; PP_00007; phage(gi100002) 4.34e-73 8 6142..6423 PHAGE_Klebsi_2146_NC_049472: hypothetical protein; PP_00008; phage(gi100003) 1.03e-63 9 6521..7138 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00009; phage(gi100201) 1.18e-152 10 7138..7776 PHAGE_Escher_vB_EcoM_KWBSE43_6_NC_048186: hypothetical protein; PP_00010; phage(gi100006) 1.28e-154 11 7885..8160 PHAGE_Klebsi_2146_NC_049472: hypothetical protein; PP_00011; phage(gi100006) 1.70e-61 12 8237..10615 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00012; phage(gi100204) 0.0 13 10,669..11235 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00013; phage(gi100205) 4.12e-135 14 11,299..11625 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: ribonucleoside diphosphate reductase large subunit; PP_00014; phage(gi100207) 2.00e-75 15 11,701..12309 PHAGE_Escher_vB_EcoM_KWBSE43_6_NC_048186: hypothetical protein; PP_00015; phage(gi100014) 2.54e-152 16 12,320..12595 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00016; phage(gi100003) 1.98e-61 17 12,592..13656 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00017; phage(gi100004) 0.0 18 13,656..13868 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00018; phage(gi100005) 1.50e-46 19 14,051..14539 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00019; phage(gi100006) 4.28e-107 20 14,594..14782 PHAGE_Salmon_maane_NC_049508: hypothetical protein; PP_00020; phage(gi100091) 4.00e-39 21 14,779..15120 PHAGE_Pseudo_pf16_NC_041881: hypothetical protein; PP_00021; phage(gi100022) 1.36e-09 22 15,192..15986 PHAGE_Shigel_MK_13_NC_049455: hypothetical protein; PP_00022; phage(gi100191) 0.0 23 16,021..16728 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00023; phage(gi100010) 4.52e-174 24 16,774..17616 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00024; phage(gi100011) 0.0 25 17,704..19986 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00025; phage(gi100012) 0.0 26 20,061..21164 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00026; phage(gi100013) 0.0 27 21,174..21398 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00027; phage(gi100014) 4.91e-48 28 21,493..21948 PHAGE_Escher_vB_EcoM_KWBSE43_6_NC_048186: hypothetical protein; PP_00028; phage(gi100028) 2.45e-109 29 21,955..22323 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00029; phage(gi100016) 5.01e-87 30 complement(22,306..22686) PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00030; phage(gi100017) 2.50e-89 31 complement(22,750..24360) PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00031; phage(gi100018) 0.0 32 complement(24,861..25664) PHAGE_Klebsi_2146_NC_049472: hypothetical protein; PP_00032; phage(gi100030) 0.0 33 25,714..26244 PHAGE_Klebsi_2146_NC_049472: hypothetical protein; PP_00033; phage(gi100031) 1.83e-129 34 26,216..26704 PHAGE_Klebsi_2146_NC_049472: u-spanin; PP_00034; phage(gi100032) 2.31e-116 35 26,743..27354 PHAGE_Klebsi_2146_NC_049472: endolysin; PP_00035; phage(gi100033) 2.45e-152 36 27,358..27486 hypothetical; PP_00036 0.0 37 27,630..27875 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00037; phage(gi100024) 1.19e-54 38 27,868..28110 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00038; phage(gi100025) 4.04e-51 39 28,120..28359 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00039; phage(gi100026) 3.57e-53 40 28,456..29502 PHAGE_Klebsi_2146_NC_049472: hypothetical protein; PP_00040; phage(gi100038) 0.0 41 29,545..29949 PHAGE_Klebsi_2146_NC_049472: hypothetical protein; PP_00041; phage(gi100039) 6.10e-87 42 complement(29,973..30920) PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00042; phage(gi100028) 0.0 43 30,974..31681 PHAGE_Klebsi_2146_NC_049472: DNA adenine methyltransferase; PP_00043; phage(gi100041) 1.77e-175 44 31,748..32482 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00044; phage(gi100030) 2.21e-178 45 32,505..32816 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00045; phage(gi100031) 1.26e-71 46 32,955..33866 PHAGE_Klebsi_2146_NC_049472: DNA helicase; PP_00046; phage(gi100044) 0.0 47 33,941..34603 PHAGE_Klebsi_2146_NC_049472: putative transcriptional regulator; PP_00047; phage(gi100045) 1.36e-164 48 34,603..35646 PHAGE_Klebsi_2146_NC_049472: HNH endonuclease; PP_00048; phage(gi100046) 0.0 49 35,643..36212 PHAGE_Escher_vB_EcoM_KWBSE43_6_NC_048186: recombination protein; PP_00049; phage(gi100049) 3.94e-139 50 36,209..36760 PHAGE_Escher_vB_EcoM_KWBSE43_6_NC_048186: putative single-stranded DNA binding protein; PP_00050; phage(gi100050) 1.53e-135 51 36,760..37278 PHAGE_Klebsi_2146_NC_049472: recombination protein; PP_00051; phage(gi100049) 1.22e-123 52 37,263..38348 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00052; phage(gi100038) 0.0 53 38,326..38655 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00053; phage(gi100039) 6.14e-75 54 38,662..40089 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00054; phage(gi100040) 0.0 55 40,152..40487 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: DNA adenine methyltransferase; PP_00055; phage(gi100041) 2.35e-79 56 40,501..40815 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00056; phage(gi100042) 9.10e-71 57 40,934..42127 PHAGE_Klebsi_2146_NC_049472: minor tail protein; PP_00057; phage(gi100055) 0.0 58 42,124..42264 PHAGE_Klebsi_2146_NC_049472: minor tail protein; PP_00058; phage(gi100056) 1.21e-23 59 42,264..42476 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: putative transcriptional regulator; PP_00059; phage(gi100045) 3.95e-46 60 42,478..42666 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: HNH endonuclease; PP_00060; phage(gi100046) 1.74e-37 61 42,668..43009 PHAGE_Escher_vB_EcoM_KWBSE43_6_NC_048186: hypothetical protein; PP_00061; phage(gi100061) 2.78e-78 62 43,067..43675 PHAGE_Klebsi_2146_NC_049472: hypothetical protein; PP_00062; phage(gi100061) 2.98e-137 63 43,717..44274 PHAGE_Klebsi_2146_NC_049472: hypothetical protein; PP_00063; phage(gi100062) 1.10e-122 64 44,528..45949 PHAGE_Klebsi_2146_NC_049472: hypothetical protein; PP_00064; phage(gi100064) 0.0 65 45,946..46410 PHAGE_Entero_EspM4VN_NC_049384: hypothetical protein; PP_00065; phage(gi100018) 2.03e-33 66 46,410..46667 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: exonuclease; PP_00066; phage(gi100048) 2.15e-54 67 46,633..46893 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: recombination protein; PP_00067; phage(gi100049) 9.74e-58 68 46,883..47545 PHAGE_Klebsi_2146_NC_049472: capsid maturation protease; PP_00068; phage(gi100067) 6.11e-163 69 complement(47,548..49533) PHAGE_Klebsi_2146_NC_049472: head morphogenesis protein; PP_00069; phage(gi100068) 0.0 70 complement(49,544..50932) PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: tail fiber protein; PP_00070; phage(gi100052) 0.0 71 complement(50,929..51486) PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: tail assembly protein; PP_00071; phage(gi100053) 1.27e-134 72 complement(51,501..52469) PHAGE_Klebsi_2146_NC_049472: terminase small subunit; PP_00072; phage(gi100071) 0.0 73 52,523..53140 PHAGE_Klebsi_2146_NC_049472: hypothetical protein; PP_00073; phage(gi100072) 4.03e-151 74 complement(53,148..53345) PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: tail length tape-measure protein; PP_00074; phage(gi100057) 1.82e-41 75 complement(53,348..53755) PHAGE_Klebsi_2146_NC_049472: hypothetical protein; PP_00075; phage(gi100074) 4.21e-93 76 complement(53,766..54272) PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00076; phage(gi100059) 8.08e-125 77 complement(54,265..54609) PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00077; phage(gi100060) 3.93e-77 78 complement(54,672..55067) PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00078; phage(gi100061) 3.06e-95 79 complement(55,064..55435) PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00079; phage(gi100062) 3.67e-88 80 complement(55,500..55796) PHAGE_Klebsi_2146_NC_049472: hypothetical protein; PP_00080; phage(gi100083) 7.52e-68 81 complement(55,796..56425) PHAGE_Escher_vB_EcoM_KWBSE43_6_NC_048186: hypothetical protein; PP_00081; phage(gi100083) 3.01e-154 82 complement(56,418..57023) PHAGE_Klebsi_2146_NC_049472: hypothetical protein; PP_00082; phage(gi100085) 1.74e-147 83 complement(57,020..57247) PHAGE_Klebsi_2146_NC_049472: hypothetical protein; PP_00083; phage(gi100086) 1.74e-49 84 complement(57,247..57366) PHAGE_Escher_vB_EcoM_KWBSE43_6_NC_048186: hypothetical protein; PP_00084; phage(gi100086) 5.79e-20 85 complement(57,410..57814) PHAGE_Klebsi_2146_NC_049472: hypothetical protein; PP_00085; phage(gi100088) 7.03e-95 86 complement(57,818..58132) PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: head morphogenesis protein; PP_00086; phage(gi100068) 4.24e-73 87 complement(58,132..58422) PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: portal protein; PP_00087; phage(gi100069) 2.06e-63 88 complement(58,465..59796) PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: terminase large subunit; PP_00088; phage(gi100070) 0.0 89 complement(59,798..61708) PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: terminase small subunit; PP_00089; phage(gi100071) 0.0 90 complement(61,757..62383) PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00090; phage(gi100072) 1.30e-156 91 complement(62,380..62865) PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00091; phage(gi100073) 3.06e-117 92 complement(63,147..63650) PHAGE_Escher_vB_EcoM_KWBSE43_6_NC_048186: hypothetical protein; PP_00092; phage(gi100095) 6.72e-107 93 complement(63,739..63960) PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00093; phage(gi100075) 2.76e-45 94 complement(63,962..64771) PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00094; phage(gi100076) 0.0 95 complement(64,750..64929) PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00095; phage(gi100077) 4.21e-38 96 complement(65,174..65584) PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: single-stranded-DNA-specific exonuclease; PP_00096; phage(gi100078) 1.59e-93 97 complement(65,562..65858) PHAGE_Klebsi_2146_NC_049472: putative DNA polymerase; PP_00097; phage(gi100100) 3.54e-67 98 complement(65,911..67497) PHAGE_Klebsi_2146_NC_049472: nicotinamide mononucleotide transporter; PP_00098; phage(gi100101) 0.0 99 complement(67,531..70287) PHAGE_Klebsi_2146_NC_049472: hypothetical protein; PP_00099; phage(gi100102) 0.0 100 complement(70,390..70728) PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: deoxynucleoside kinase; PP_00100; phage(gi100082) 1.23e-76 101 complement(70,739..70906) PHAGE_Klebsi_Menlow_NC_047901: hypothetical protein; PP_00101; phage(gi100006) 5.62e-33 102 complement(70,903..71121) PHAGE_Klebsi_Magnus_NC_049462: hypothetical protein; PP_00102; phage(gi100006) 4.93e-46 103 complement(71,121..71774) PHAGE_Klebsi_Magnus_NC_049462: hypothetical protein; PP_00103; phage(gi100008) 2.16e-147 104 complement(71,771..72157) PHAGE_Klebsi_Magnus_NC_049462: hypothetical protein; PP_00104; phage(gi100010) 4.07e-88 105 complement(72,223..72657) PHAGE_Klebsi_Magnus_NC_049462: hypothetical protein; PP_00105; phage(gi100012) 6.27e-99 106 complement(72,662..73225) PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00106; phage(gi100088) 1.17e-135 107 complement(73,242..74333) PHAGE_Klebsi_Menlow_NC_047901: hypothetical protein; PP_00107; phage(gi100018) 0.0 108 complement(74,330..74449) PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: putative HNH homing endonuclease; PP_00108; phage(gi100090) 2.35e-20 109 complement(74,644..75861) PHAGE_Klebsi_Magnus_NC_049462: hypothetical protein; PP_00109; phage(gi100020) 0.0 110 complement(75,865..76245) PHAGE_Klebsi_2146_NC_049472: hypothetical protein; PP_00110; phage(gi100113) 4.87e-89 111 complement(76,242..76445) PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00111; phage(gi100093) 3.11e-42 112 complement(76,448..77332) PHAGE_Klebsi_Menlow_NC_047901: hypothetical protein; PP_00112; phage(gi100028) 0.0 113 complement(77,332..77580) PHAGE_Klebsi_Menlow_NC_047901: hypothetical protein; PP_00113; phage(gi100030) 1.56e-54 114 complement(77,642..78433) PHAGE_Klebsi_Menlow_NC_047901: u-spanin; PP_00114; phage(gi100032) 0.0 115 complement(78,430..78780) PHAGE_Klebsi_2146_NC_049472: hypothetical protein; PP_00115; phage(gi100118) 6.15e-81 116 complement(78,847..81843) PHAGE_Klebsi_Magnus_NC_049462: holin; PP_00116; phage(gi100034) 0.0 117 81,943..82494 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00117; phage(gi100099) 5.77e-130 118 82,841..83065 PHAGE_Klebsi_Menlow_NC_047901: hypothetical protein; PP_00118; phage(gi100042) 5.91e-50 119 83,134..83661 PHAGE_Klebsi_2146_NC_049472: DNA polymerase III alpha subunit; PP_00119; phage(gi100121) 1.13e-127 120 complement(83,743..84195) PHAGE_Klebsi_Menlow_NC_047901: HNH endonuclease; PP_00120; phage(gi100046) 1.36e-101 121 complement(84,228..84686) PHAGE_Klebsi_Menlow_NC_047901: exonuclease; PP_00121; phage(gi100048) 7.86e-109 122 complement(84,727..84879) PHAGE_Klebsi_Menlow_NC_047901: putative single-stranded DNA binding protein; PP_00122; phage(gi100050) 2.86e-29 123 complement(84,876..86246) PHAGE_Escher_vB_EcoM_KWBSE43_6_NC_048186: HNH homing endonuclease; PP_00123; phage(gi100129) 0.0 124 complement(86,316..86921) PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00124; phage(gi100104) 6.33e-147 125 complement(87,247..87320) tRNA 0.0 126 complement(87,443..87526) tRNA 0.0 127 complement(88,112..88187) tRNA 0.0 128 complement(88,198..88273) tRNA 0.0 129 complement(88,281..88472) PHAGE_Salmon_rabagast_NC_049499: hypothetical protein; PP_00125; phage(gi100191) 1.91e-38 130 complement(91,814..92383) PHAGE_Klebsi_0507_KN2_1_NC_022343: hypothetical protein; PP_00126; phage(gi543171769) 3.59e-140 131 92,738..94519 PHAGE_Klebsi_0507_KN2_1_NC_022343: baseplate wedge subunit; PP_00127; phage(gi543171770) 0.0 132 94,503..95354 PHAGE_Klebsi_2146_NC_049472: putative baseplate hub protein; PP_00128; phage(gi100137) 0.0 133 95,359..96684 PHAGE_Klebsi_0507_KN2_1_NC_022343: hypothetical protein; PP_00129; phage(gi543171772) 0.0 134 96,736..99618 PHAGE_Klebsi_0507_KN2_1_NC_022343: putative tailspike protein; PP_00130; phage(gi543171773) 0.0 135 99,630..99902 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00131; phage(gi100119) 2.80e-46 136 99,949..101295 PHAGE_Klebsi_0507_KN2_1_NC_022343: hypothetical protein RaK2_00525; PP_00132; phage(gi543171777) 9.31e-53 137 101,353..104010 PHAGE_Klebsi_0507_KN2_1_NC_022343: putative tail fiber protein; PP_00133; phage(gi543171774) 6.23e-106 138 104,080..106164 PHAGE_Klebsi_0507_KN2_1_NC_022343: tail spike protein head-binding protein; PP_00134; phage(gi543171775) 1.21e-29 139 106,379..108619 PHAGE_Klebsi_2146_NC_049472: major tail protein; PP_00135; phage(gi100145) 6.91e-33 140 108,630..108905 PHAGE_Erwini_PEp14_NC_016767: hypothetical protein; PP_00136; phage(gi374531865) 3.26e-10 141 109,001..113839 PHAGE_Klebsi_0507_KN2_1_NC_022343: vrlC protein; PP_00137; phage(gi543171778) 0.0 142 113,890..114138 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: recombination endonuclease subunit D12; PP_00138; phage(gi100127) 1.97e-54 143 114,122..114460 PHAGE_Klebsi_0507_KN2_1_NC_022343: hypothetical protein; PP_00139; phage(gi543171779) 4.82e-74 144 114,447..115199 PHAGE_Klebsi_2146_NC_049472: prohead protease; PP_00140; phage(gi100150) 0.0 145 complement(115,228..115440) PHAGE_Klebsi_Magnus_NC_049462: putative methyltransferase; PP_00141; phage(gi100107) 6.27e-45 146 115,502..116143 PHAGE_Klebsi_0507_KN2_1_NC_022343: neck protein; PP_00142; phage(gi543171781) 8.09e-156 147 116,146..116844 PHAGE_Klebsi_0507_KN2_1_NC_022343: proximal tail sheath stabilization; PP_00143; phage(gi543171782) 1.50e-175 148 116,847..117533 PHAGE_Klebsi_0507_KN2_1_NC_022343: terminase DNA packaging enzyme small subunit; PP_00144; phage(gi543171783) 5.73e-164 149 117,514..119730 PHAGE_Klebsi_0507_KN2_1_NC_022343: terminase subunit for DNA packaging, nuclease and ATPase; PP_00145; phage(gi543171784) 0.0 150 119,776..121671 PHAGE_Klebsi_0507_KN2_1_NC_022343: tail sheath protein; PP_00146; phage(gi543171785) 0.0 151 121,740..122195 PHAGE_Klebsi_0507_KN2_1_NC_022343: GIY-YIG endonuclease; PP_00147; phage(gi543171786) 8.88e-109 152 122,230..122763 PHAGE_Klebsi_0507_KN2_1_NC_022343: tail tube protein; PP_00148; phage(gi543171787) 9.75e-129 153 122,832..124514 PHAGE_Klebsi_0507_KN2_1_NC_022343: portal vertex protein of the head; PP_00149; phage(gi543171788) 0.0 154 124,560..124724 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00150; phage(gi100140) 7.75e-31 155 124,734..125042 PHAGE_Klebsi_0507_KN2_1_NC_022343: putative prohead core protein; PP_00151; phage(gi543171789) 1.65e-65 156 125,053..125718 PHAGE_Klebsi_0507_KN2_1_NC_022343: putative prohead protease; PP_00152; phage(gi543171790) 6.09e-164 157 125,764..126618 PHAGE_Klebsi_0507_KN2_1_NC_022343: prohead core scaffold protein; PP_00153; phage(gi543171791) 0.0 158 126,713..128035 PHAGE_Klebsi_0507_KN2_1_NC_022343: phage major head protein/major capsid protein; PP_00154; phage(gi543171792) 0.0 159 128,119..128766 PHAGE_Salmon_SS9_NC_049458: hypothetical protein; PP_00155; phage(gi100178) 5.46e-162 160 128,826..129071 PHAGE_Klebsi_Menlow_NC_047901: pore-forming tail tip protein; PP_00156; phage(gi100139) 6.43e-54 161 129,068..129319 PHAGE_Klebsi_Magnus_NC_049462: pore-forming tail tip protein; PP_00157; phage(gi100139) 2.20e-39 162 129,742..129948 PHAGE_Klebsi_Menlow_NC_047901: major tail protein; PP_00158; phage(gi100145) 1.32e-41 163 129,929..130159 PHAGE_Klebsi_0507_KN2_1_NC_022343: hypothetical protein; PP_00159; phage(gi543171793) 4.17e-48 164 130,175..130612 PHAGE_Klebsi_2146_NC_049472: tail fibers protein; PP_00160; phage(gi100168) 1.83e-105 165 130,612..131052 PHAGE_Klebsi_0507_KN2_1_NC_022343: hypothetical protein; PP_00161; phage(gi543171795) 1.30e-102 166 131,054..131290 PHAGE_Klebsi_Menlow_NC_047901: portal protein; PP_00162; phage(gi100153) 6.57e-53 167 131,394..131693 PHAGE_Klebsi_2146_NC_049472: tail fibers protein; PP_00163; phage(gi100171) 1.47e-68 168 132,038..132481 PHAGE_Klebsi_0507_KN2_1_NC_022343: hypothetical protein; PP_00164; phage(gi543171796) 5.20e-105 169 132,505..132672 PHAGE_Klebsi_2146_NC_049472: baseplate wedge; PP_00165; phage(gi100174) 7.61e-33 170 132,707..133435 PHAGE_Klebsi_Magnus_NC_049462: hypothetical protein; PP_00166; phage(gi100163) 8.06e-176 171 complement(133,436..134092) PHAGE_Klebsi_0507_KN2_1_NC_022343: hypothetical protein; PP_00167; phage(gi543171798) 5.48e-154 172 134,122..134622 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00168; phage(gi100160) 4.67e-118 173 134,660..135124 PHAGE_Klebsi_0507_KN2_1_NC_022343: putative DNA repair/recombination protein UvsY; PP_00169; phage(gi543171800) 4.48e-109 174 135,124..135870 PHAGE_Klebsi_Magnus_NC_049462: tail fibers protein; PP_00170; phage(gi100171) 0.0 175 135,898..137415 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: D5 protein; PP_00171; phage(gi100164) 0.0 176 complement(137,400..137783) PHAGE_Klebsi_2146_NC_049472: hypothetical protein; PP_00172; phage(gi100181) 4.65e-89 177 138,107..138775 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00173; phage(gi100166) 3.32e-164 178 138,861..139850 PHAGE_Klebsi_0507_KN2_1_NC_022343: sliding clamp holder; PP_00174; phage(gi543171805) 0.0 179 139,853..140275 PHAGE_Klebsi_0507_KN2_1_NC_022343: clamp holder for DNA polymerase; PP_00175; phage(gi543171806) 1.45e-100 180 140,304..140768 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00176; phage(gi100169) 2.11e-112 181 140,785..141651 PHAGE_Klebsi_0507_KN2_1_NC_022343: hypothetical protein; PP_00177; phage(gi543171808) 0.0 182 141,723..142943 PHAGE_Klebsi_0507_KN2_1_NC_022343: hypothetical protein; PP_00178; phage(gi543171809) 0.0 183 143,281..145317 PHAGE_Klebsi_0507_KN2_1_NC_022343: hypothetical protein; PP_00179; phage(gi543171810) 0.0 184 145,354..145728 PHAGE_Klebsi_0507_KN2_1_NC_022343: hypothetical protein; PP_00180; phage(gi543171811) 2.23e-78 185 145,794..146546 PHAGE_Klebsi_0507_KN2_1_NC_022343: hypothetical protein; PP_00181; phage(gi543171815) 7.76e-177 186 146,611..146967 PHAGE_Klebsi_0507_KN2_1_NC_022343: hypothetical protein; PP_00182; phage(gi543171816) 3.73e-78 187 146,975..147118 PHAGE_Klebsi_Magnus_NC_049462: hypothetical protein; PP_00183; phage(gi100205) 1.62e-25 188 147,111..149309 PHAGE_Klebsi_0507_KN2_1_NC_022343: hypothetical protein; PP_00184; phage(gi543171817) 0.0 189 149,354..149677 PHAGE_Klebsi_0507_KN2_1_NC_022343: putative acyl carrier protein; PP_00185; phage(gi543171818) 3.82e-70 190 149,992..150228 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: hypothetical protein; PP_00186; phage(gi100180) 2.24e-48 191 150,283..150651 PHAGE_Klebsi_0507_KN2_1_NC_022343: hypothetical protein; PP_00187; phage(gi543171819) 2.91e-85 192 150,671..150850 PHAGE_Klebsi_vB_KpnM_KpS110_NC_047932: ATP-dependent Clp protease; PP_00188; phage(gi100182) 2.20e-34 193 150,975..151424 PHAGE_Klebsi_0507_KN2_1_NC_022343: phage associated protein; PP_00189; phage(gi543171821) 2.45e-107 194 151,503..152189 PHAGE_Klebsi_0507_KN2_1_NC_022343: hypothetical protein; PP_00190; phage(gi543171822) 2.05e-168 195 152,189..152533 PHAGE_Klebsi_0507_KN2_1_NC_022343: hypothetical protein; PP_00191; phage(gi543171823) 4.93e-80 196 152,583..153323 PHAGE_Klebsi_0507_KN2_1_NC_022343: hypothetical protein; PP_00192; phage(gi543171824) 0.0 197 153,372..154079 PHAGE_Klebsi_0507_KN2_1_NC_022343: ImpD; PP_00193; phage(gi543171825) 2.96e-177 198 154,076..154351 PHAGE_Klebsi_Menlow_NC_047901: hypothetical protein; PP_00194; phage(gi100233) 1.15e-60 199 154,332..154658 PHAGE_Serrat_vB_Sru_IME250_NC_042047: tail fibers protein; PP_00195; phage(gi100168) 1.18e-61 200 154,655..154969 PHAGE_Klebsi_Menlow_NC_047901: hypothetical protein; PP_00196; phage(gi100237) 1.06e-71 201 complement(154,997..155113) PHAGE_Klebsi_0507_KN2_1_NC_022343: hypothetical protein; PP_00198; phage(gi543171827) 3.12e-20 202 155,087..155365 PHAGE_Klebsi_Menlow_NC_047901: NAD dependent DNA ligase subunit A; PP_00197; phage(gi100239) 2.02e-59 In the table legend, The "ORF" column denotes the Open Reading Frame (ORF) number, serving as a unique identifier for each ORF. The "CDS Position" column indicates the position of the coding sequence (CDS) within the genome. In the "BLAST Hit" column, annotations or descriptions of sequence similarities identified through BLAST are provided, often specifying the source organism and the function or predicted function of the sequence. The "E-Value" column presents the Expect value (E-value), a parameter quantifying the statistical significance of sequence similarity. A lower E-value indicates a more significant match, with the number of expected chance hits decreasing accordingly Fig. 5 Whole genome map of phage Zag1. The figure displays the whole genome map of phage Zag1. The circular representation showcases the phage's genomic sequence, indicating the positions of various ORFs and functional elements. The map highlights key features and regions of interest within the phage's genetic structure Fig. 6 Phylogenetic tree depicting the evolutionary relationships among various bacteriophages, including Klebsiella phage cp16, cp21, P01, KP6, Menlow, PWKp5, vB_KpnM-20, vB_KpnM_KpS110, vB_KpnS_MDA2066, UPM 2146, vB_KqM-Westerburg, vB_KqM-LilBean, and vB_KqM-Bilbo, as well as Escherichia phage vB_EcoM_KWBSE43-6, Klebsiella phage T751, Klebsiella phage 0507-KN2-1 DNA, and KZag1. The scale bar represents a genetic distance corresponding to 0.01 substitutions per site, indicating minimal evolutionary divergence among the depicted phages Annotation table CDS Position of KZag1 In the table legend, The "ORF" column denotes the Open Reading Frame (ORF) number, serving as a unique identifier for each ORF. The "CDS Position" column indicates the position of the coding sequence (CDS) within the genome. In the "BLAST Hit" column, annotations or descriptions of sequence similarities identified through BLAST are provided, often specifying the source organism and the function or predicted function of the sequence. The "E-Value" column presents the Expect value (E-value), a parameter quantifying the statistical significance of sequence similarity. A lower E-value indicates a more significant match, with the number of expected chance hits decreasing accordingly Whole genome map of phage Zag1. The figure displays the whole genome map of phage Zag1. The circular representation showcases the phage's genomic sequence, indicating the positions of various ORFs and functional elements. The map highlights key features and regions of interest within the phage's genetic structure Phylogenetic tree depicting the evolutionary relationships among various bacteriophages, including Klebsiella phage cp16, cp21, P01, KP6, Menlow, PWKp5, vB_KpnM-20, vB_KpnM_KpS110, vB_KpnS_MDA2066, UPM 2146, vB_KqM-Westerburg, vB_KqM-LilBean, and vB_KqM-Bilbo, as well as Escherichia phage vB_EcoM_KWBSE43-6, Klebsiella phage T751, Klebsiella phage 0507-KN2-1 DNA, and KZag1. The scale bar represents a genetic distance corresponding to 0.01 substitutions per site, indicating minimal evolutionary divergence among the depicted phages

Clinical specimens suspected to contain Klebsiella isolates were collected from the sputum of patients at Zagazig University Hospital, Sharqia, Egypt, following the acquisition of informed consent. The collected samples were promptly transported to the laboratory for further processing. To ensure the safety of laboratory personnel and prevent cross-contamination, the samples were handled in accordance with standard biosafety protocols. Laboratory personnel wore appropriate personal protective equipment (PPE), including gloves and laboratory coats, during sample processing. Serial dilutions of the collected samples were performed to achieve a suitable bacterial load for isolation. Using a sterile loop or spreader, the diluted samples were streaked onto selective agar plates specifically designed for the isolation of Klebsiella , such as MacConkey agar or blood agar (Oxoid, United Kingdom) [ 16 ]. These plates were then incubated at the optimal temperature for Klebsiella growth, typically 35–37°C, for a period of 24–48 h. Following incubation, the agar plates were examined for the presence of bacterial colonies. Colonies displaying characteristic Klebsiella morphology, such as a mucoid appearance, lactose fermentation, and non-hemolytic on blood agar plates, was selected. Using a sterile loop, these selected colonies were streaked onto fresh agar plates to obtain pure cultures, ensuring the isolation of individual Klebsiella strains [ 17 ]. The isolated colonies were then incubated under appropriate conditions to facilitate further growth and identification. To confirm the identity of the isolated Klebsiella strains, additional tests were conducted. Specifically, DNA extraction was conducted using the QIAamp DNA Mini Kit supplied from QIAGEN (USA) with Catalogue no. 51304. Subsequently, 16S rRNA sequencing was performed using the ready reaction Bigdye Terminator V3.1 cycle sequencing kit (Perkin-Elmer/Applied Biosystems, Foster City, CA), with Cat. No. 4336817, provided by Sigma Company, located in Giza, Egypt, for the accurate identification of the Klebsiella strains [ 18 ]. The GenBank accession number link provided: https://www.ncbi.nlm.nih.gov/nucleotide/OP942216.1 . The antibiotic susceptibility test was conducted on the clinical isolates to determine the susceptibility pattern of bacteria. The antibiotic sensitivity test was conducted using the Kirby-Bauer method (also called the disc diffusion test) on Muller-Hinton agar [ 12 ]. Sterilized Müller-Hinton agar (Oxoid, USA) was poured into Petri dishes in 10 mL aliquots. Once the agar solidified, 100 µL aliquots of broth cultures, inoculated with 10 8 CFU mL −1 of the tested bacterial strains, were evenly spread over the surface of the agar plates. Antibiotic discs were carefully placed on the plate surfaces. Fifteen bacterial isolates were tested against the following antibiotics: Imipenem (IPM) (10 μg), Polymyxin B (PB) (300 μg), Amikacin (AK) (30 μg), Erythromycin (E) (15 μg), Trimethoprim/sulphamethoxazole (SXT) (25 μg), Cefoxitin (FOX) (30μg), Cefepime (FEP) (30μg), Ceftriaxone (CRO) (30μg), Ceftazidime (CAZ) (30μg), Gentamicin (CN) (10μg), Ciprofloxacin (CIP) (5μg), Erythromycin (E) (15μg), Chloramphenicol (C) (30mcg), Piperacillin (PRL) (100 μg), and Tetracycline (TE) (30 μg). The plates were then incubated upside down at 37°C for 16–18 h. The diameter of the inhibition zone around each antibiotic disc was measured in millimeters (mm). The interpretation of the inhibition zone diameter was classified as sensitive (S), intermediate (I), or resistant (R) based on the interpretative criteria recommended by CLSI [ 19 ] for antimicrobial susceptibility testing. Additionally, the multiple antibiotic resistances (MAR) index was calculated to determine the level of antibiotic resistance. The MAR index is the ratio between the number of antibiotics to which the bacteria are resistant and the total number of antibiotics used, as described by Sayah et al. [ 20 ]. Bacteriophages were isolated from different sewage water samples obtained from Sharkia Governorate, Egypt, using the enrichment technique [ 21 ] . Initially, 100 mL of sewage was filtered through a 0.45μm-filter membrane and combined with an equal volume of nutrient broth in 500 mL Erlenmeyer flasks. Subsequently, 5 mL of fresh K. pneumoniae culture (2.0 × 10 8 CFU mL −1 ) was added to each sewage sample. The flasks were incubated on a shaker (120 rpm) at 37 ºC for 24 h. After incubation, the mixture was centrifuged at 10,000 rpm for 20 min, and the resulting supernatant was filtered through a 0.45μm-filter membrane to detect the presence of phages using spot test and plaque assay methods with K. pneumoniae as the host. Phage titration was performed by tenfold serial dilution of the samples in saline solution and spotting them on lawns of K. pneumoniae , with the phage titer expressed in plaque forming units (PFU) per mL. Phages were propagated and purified from various single-plaque isolates following Kim et al. [ 21 ]. The isolated phages underwent five successive single-plaque isolations until homogenous plaques were obtained. In each isolation, a single plaque was picked and incubated with 1 mL of nutrient broth containing an overnight culture of K. pneumoniae at 37 ºC with agitation. After incubation, the phage-host mixture was centrifuged at 10,000 rpm for 10 min, and the supernatants were filtered through a 0.45μm Millipore filter to eliminate any bacterial contamination. The purified phages were stored at 4ºC for further characterization. The morphology and structure of the isolated bacteriophage were analyzed using transmission electron microscopy (TEM), according to the method described by Abdel-Haliem and Askora [ 22 ].The phage suspension was prepared, containing a concentration of 10 8 (PFU mL −1 ). A small volume of the phage suspension was applied onto Carbon-coated formvar films on 200 mesh copper grids. Excess liquid was carefully removed from the grid using filter paper. A few drops of the Sodium phosphotungstate solution were added onto the grid, covering the phage particles. Excess Sodium phosphotungstate solution was gently removed from the grid using filter paper. Images of the phage particles were captured using a Hitachi H600A electron microscope at the Faculty of Agriculture, Mansoura University, Egypt. The one-step growth curve of the phage was determined following a standard protocol [ 16 ] . Briefly, a culture of the host bacterium K. pneumoniae was grown to the logarithmic phase of growth. The bacterial culture was then infected with the phage at a multiplicity of infection (MOI) of 0.01, which means that for every bacterium, only one phage was added. After allowing the phage to adsorb to the bacterial cells for a specific duration 5 min, the mixture was diluted and plated onto agar plates to determine the number of viable phages (PFU) at time zero. This represented the initial phage count. Subsequently, samples were taken at regular intervals every 5 min for certain duration 2 h. Each sample was immediately diluted and plated onto agar plates to quantify the number of infective phages at each time point. This allowed for the construction of the growth curve. To calculate the burst size, the number of new phages released from each infected bacterium was determined by comparing the phage count at the peak of the growth curve with the initial phage count. The burst size represents the average number of phages released per infected bacterium. The experiment was performed in triplicate to ensure the reliability of the results, and the average values along with standard deviations were reported. The host range of Kzag1 phage was determined experimentally using a collection of bacterial strains, including K. pneumoniae and related species, as potential hosts. Each bacterial strain was cultured to the logarithmic growth phase, and 100 µL of each culture was spread on separate agar plates. After drying, small drops of the phage suspension were added to the agar surface to allow phage attachment to the bacterial cells. The plates were then incubated at the optimal growth temperature for the bacterial strains at 37°C. After an appropriate incubation period 18–24 h, the plates were inspected for the presence of plaques, indicating successful phage infection and subsequent bacterial cell lysis. The absence of plaques indicated that the phage could not infect the specific bacterial strain. The influence of temperature and pH on the stability of the phage was examined. Thermal stability tests were performed following the protocol described by Mahmoud et al. [ 23 ]. Phage samples with a known titer (10 6 –10 8 PFU mL −1 ) were exposed to different temperatures ranging from 30°C to 100°C for 10-min intervals in a water bath incubator. The infectivity of the phages was determined immediately after incubation using the double-layer agar plate method. Additionally, pH stability tests were conducted by inoculating a known phage suspension (10 6 –10 8 PFU mL −1 ) into LB liquid medium obtained from Thermo Fisher Scientific, United States with pH values ranging from 3.0 to 12.0, followed by overnight incubation at 4°C. The viability of the bacteriophages was assessed using the overlay method as described by Adams [ 21 ] . The impact of Kzag1 phage on the growth of K. pneumoniae was investigated. A culture of K. pneumoniae was diluted to a final density of 2.0 × 10 8 CFU mL −1 and placed in a nutrient broth, then incubated at 37°C for 24 h. The phage suspension was also diluted to achieve a MOI of 0.1. Subsequently, 100 µL of various phage concentrations was added to the bacterial suspension [ 24 ], and the mixture was incubated under sterile conditions. The survival of K. pneumoniae was assessed at intervals of 5, 10, 15, 20, 25, 30, and 35 min using the plaque assay method. To assess the impact of Zag1 phage on biofilm formation by Klebsiella , the following experimental procedures were conducted according to Jamal et al. [ 25 ]. A biofilm was developed by introducing 200 μL of bacterial culture (10 8 CFU mL −1 ) into each well of a 96-well flat-bottomed polystyrene microtiter plate, followed by incubation at 37°C for 24 h with gentle agitation at 120 rpm. After the biofilm formation period, any excess fluid was discarded, and the wells were washed twice with 0.9% NaCl to eliminate unattached planktonic cells. The plate was then allowed to dry at 37°C for one hour. The isolated phages were diluted in 0.9% NaCl and added to the respective wells containing their host bacteria. Different concentrations of the Zag1phage was used, including MOI = 0.1, MOI = 1, and MOI = 5. The control wells were set up using uninoculated normal saline, and they remained untreated throughout the experiment. Specifically, the uninoculated normal saline serves as the negative control, while the well inoculated with bacterial culture serves as the positive control. The plates were incubated at 37°C with constant shaking at 120 rpm for an additional day after the establishment of the biofilm. Excess fluid was removed from each well, and the biofilms were washed as previously described. The wells were left to dry for one hour at 37°C. To determine the total biomass of the biofilm, staining with 1% crystal violet was performed for 20 min. The plates were then washed with distilled water and air-dried. Next, 200 μl of 0.9% NaCl solution was added to each well, and the absorbance was measured at OD570 using an ELISA plate reader (Biotek Synergy HT Microplate Reader, USA. Triplicate measurements were taken for both control and test samples. The phage DNA sequencing and subsequent bioinformatics analysis were carried out as follows: Initially, phage DNA was extracted using the phenol–chloroform method, following the protocol by Sambrook and Russell [ 26 ]. The purified phage DNA was then subjected to sequencing using the Illumina Miseq platform (Illumina, San Diego, CA, United States). The obtained sequencing data was assembled using SPAdeS v.3.13.0 [ 27 ] by Sanigen Inc., South Korea. To identify ORFs, a combination of Glimmer3 [ 28 ], GeneMarkS [ 29 ], and the RAST annotation server [ 30 ] was utilized. The annotated data were organized using Artemis [ 31 ]. Furthermore, the tRNA sequence within the phage genome was analyzed using the tRNAscan-SE program. Predictions for the functions of the phage proteins were made using NCBI BLASTp and the InterProscan program [ 32 ] . The annotated genome sequence of the KZag1 phage was deposited in the NCBI GenBank database under accession number OR502445 . Phylogenetic analysis of the Zag1 phage was conducted by querying the Blast database and reconstructing a phylogenetic tree. Genomic sequences of relevant phages were retrieved from the GenBank database ( https://www.ncbi.nlm.nih.gov/genbank/ ) [ 32 ]. These sequences underwent alignment using Clustal Omega to ensure precise alignment, considering sequence homology and structural similarities [ 33 ]. Subsequently, the aligned sequences were used to construct phylogenetic tree employing the robust maximum likelihood (ML) methodology [ 34 ]. Statistical analyses were conducted to evaluate the significance of the inferred phylogenetic relationships. Each experiment was conducted in triplicate, and the average of the triplicate determinations was taken to represent the results. Statistical analyses were carried out using SPSS software package version 11.5 and Microsoft Excel 2010. The data were subjected to analysis of variance, and significant differences ( p  > 0.05) between means were determined according to Pallant [ 35 ] .

The present study focused on the analysis of a novel bacteriophage, KZag1, which specifically infects biofilms formed by K. pneumoniae . The research aimed to characterize the genome sequence and investigate the properties and potential applications of this phage. The findings contribute to our understanding of phage-host interactions and provide valuable insights for the development of phage-based therapies against K. pneumoniae biofilms. The first important aspect examined in this study was the sensitivity of K. pneumoniae strains to different antibiotics. The results revealed a high level of resistance to multiple antibiotics, indicating the presence of MDR strains. This observation aligns with previous reports highlighting the challenge of treating K. pneumoniae infections due to antibiotic resistance. The emergence of MDR strains poses a significant threat to public health, underscoring the urgent need for alternative treatment strategies [ 36 ]. To address this issue, the researchers isolated and characterized KZag1, a bacteriophage specifically targeting K. pneumoniae biofilms. The phage was successfully isolated from an enrichment culture containing K. pneumoniae , and its lytic activity against the bacterium was confirmed through spot and plaque assays. The morphological characterization using transmission electron microscopy revealed that KZag1 belongs to the Myoviridae family, possessing icosahedral heads and contractile tails. This classification provides important insights into the phage's structural characteristics, which may influence its infectivity and interaction with the host bacterium [ 37 ] . The host range analysis demonstrated that KZag1 exhibited strong lytic activity against K. pneumoniae strains but did not infect other tested bacterial strains, indicating a narrow host range. The specificity of bacteriophages towards certain bacterial hosts is a well-documented phenomenon and is influenced by various factors such as surface receptors, bacterial cell wall structures, and immune evasion mechanisms. In the case of bacteriophage KZag1, its strong lytic activity against K. pneumoniae strains while not infecting other tested bacterial strains suggests a high degree of specificity towards K. pneumoniae . This specificity can be attributed to the presence of specific receptor sites on the surface of K. pneumoniae cells that are recognized by the phage's tail fibers or other structural proteins. The absence or structural differences of these receptor sites in other bacterial species may prevent the attachment and subsequent infection by the bacteriophage. Furthermore, the genomic makeup of bacteriophage KZag1 likely contains genes encoding proteins that specifically target and interact with components unique to K. pneumoniae , contributing to its strong lytic activity against this bacterial species. While the exact mechanisms underlying the narrow host range of bacteriophage KZag1 may require further investigation, previous studies on phage-host interactions have demonstrated similar patterns of specificity, emphasizing the importance of understanding the molecular determinants driving bacteriophage infectivity. While a broad host range is desirable for therapeutic phages, narrow host specificity can still be advantageous in certain scenarios [ 38 ]. By targeting K. pneumoniae specifically, KZag1 may offer a more precise and effective approach for treating K. pneumoniae biofilm-related infections without disrupting the beneficial microbial flora. The one-step growth curve analysis provided insights into the dynamics of phage infection. The latent period, generation time, and burst size of KZag1 were determined. The relatively short latent period of 20 min followed by a rise period of 35 min suggests that the phage has a rapid replication cycle. The burst size, which ranged from 83 to 100 phages per cell, indicates the potential for efficient phage-mediated lysis of K. pneumoniae biofilms. These findings provide valuable information for optimizing the therapeutic application of KZag1, such as determining the appropriate timing and dosage for effective treatment. Phage stability is a crucial factor to consider when developing phage-based therapies [ 39 ] . The study investigated the effect of temperature and pH on the stability of KZag1. The results demonstrated that the phage remained infective and capable of lysing K. pneumoniae even after exposure to temperatures up to 60 °C, indicating its thermostability. Additionally, KZag1 exhibited optimal stability within a pH range of 6–8. However, extreme pH conditions, either highly acidic or alkaline, resulted in the loss of phage infectivity. These findings highlight the importance of considering environmental conditions when utilizing KZag1 as a therapeutic agent [ 39 ]. The characterization of KZag1 and its demonstrated activity against K. pneumoniae K9 biofilms contribute to the growing body of knowledge on phage-based therapies. By specifically targeting biofilms, which are notoriously resistant to conventional antibiotics, KZag1 offers a potential alternative for combating K. pneumoniae infections. The narrow host range of KZag1 may limit its application to K. pneumoniae strains only, but it also reduces the risk of impacting the natural microbiota. Moreover, the phage's thermostability and stability within a physiological pH range enhance its potential for practical application. The analysis of the KZag1 phage genome, with a size of approximately 157 kilobase pairs (kbp) and belonging to the Myoviridae family, revealed several intriguing findings. The presence of 202 predicted ORFs within the genome suggests a complex genetic composition with various functional elements. The results of our phylogenetic analysis shed light on the evolutionary relationships between KZag1 and Klebsiella virus 0507KN21, along with other related Klebsiella phages: Klebsiella phage T751, and Klebsiella phage cp18, suggesting a close evolutionary connection and shared genetic ancestry [ 40 ]. Such similarity in genomic organization and sequence conservation suggests common strategies for infecting and interacting with K. pneumoniae , the host bacterium. These findings offer valuable insights into the evolutionary dynamics of bacteriophages within the Klebsiella genus, crucial for understanding their evolutionary trajectories, host specificity, and potential applications in phage therapy and biotechnology. The abundance of ORFs in the KZag1 genome provides insight into the genetic diversity and complexity of the phage. These ORFs likely encode proteins involved in essential processes such as phage replication, assembly, DNA packaging, and host interaction. Detailed functional analysis of these ORFs can shed light on the mechanisms underlying the phage's lifecycle and its interaction with the host bacterium. Furthermore, the identification of specific genes associated with DNA modification, recombination, and mobile genetic elements within the KZag1 genome suggests potential mechanisms for genetic variation and adaptation. These elements may contribute to the phage's ability to evolve and overcome bacterial defense mechanisms, as well as facilitate the exchange of genetic material with other phages or bacterial hosts. The similarity to other Klebsiella phages, including Klebsiella virus 0507KN21, is particularly significant in the context of phage-based therapies [ 41 ]. The close genetic relatedness between these phages suggests that they may share similar host ranges and infection mechanisms. This similarity provides promising prospects for the development of phage cocktails or combination therapies targeting K. pneumoniae infections, including those associated with biofilms. Further studies are warranted to explore the functional significance of the identified ORFs and their role in phage-host interactions. Comparative genomics and proteomics approaches can provide insights into the shared and unique features of the related Klebsiella phages [ 42 , 43 ] . Additionally, assessing the efficacy of these phages in biofilm eradication and infection control is crucial to evaluate their potential as therapeutic agents. In conclusion this study provides valuable insights into the characterization and potential therapeutic application of KZag1, a novel bacteriophage targeting biofilms formed by K. pneumoniae . The findings contribute to our understanding of phage-host interactions and highlight the potential of phage-based therapies as an alternative to combat antibiotic-resistant infections. Further research and clinical trials are warranted to evaluate the efficacy and safety of KZag1 and its potential integration into clinical practice for the treatment of K. pneumoniae biofilm-related infections.

Klebsiella pneumoniae , a Gram-negative, non-motile, encapsulated bacterium, is a member of the Klebsiella genus within the Enterobacteriaceae family. It is responsible for various infections including pneumonia, urinary tract infections, and nosocomial infections. Respiratory tract infections caused by K. pneumoniae are widespread and severe, with a high mortality rate reaching 50% [ 1 ]. The situation is further complicated by the prevalence of antibiotic-resistant strains, with approximately 80% of the K. pneumoniae isolates being resistant to antibiotics, making treatment challenging [ 2 ]. The rise of multidrug-resistant strains, particularly those resistant to carbapenems, has become a significant issue in both hospital and community settings due to the extensive use of antibiotics and efficient transmission [ 3 ]. Additionally, K. pneumoniae is notable for its ability to form biofilms, which contributes to its significance as a major pathogen in healthcare-associated infections [ 4 ]. Biofilm formation in K. pneumoniae infections is a crucial virulence factor, facilitating its persistence in clinical settings. Biofilms provide protection against the host immune response and antibiotics, promoting the acquisition of resistance traits and the development of multidrug resistance (MDR) phenotypes. The dense extracellular matrix of biofilms physically obstructs antibiotic penetration, while metabolic changes render bacteria less susceptible to antibiotics targeting actively dividing cells. Additionally, biofilm-associated gene expression upregulates efflux pumps and stress response mechanisms, further enhancing antibiotic resistance [ 4 – 6 ]. The emergence and rapid spread of multidrug-resistant strains of K. pneumoniae have posed a significant threat to public health, highlighting the urgent need for innovative approaches to combat these infections [ 7 , 8 ]. Bacteriophages, or phages, represent a promising alternative to conventional antibiotics due to their specific targeting of bacterial pathogens [ 1 – 9 ]. These viruses have gained attention for their ability to specifically target and kill bacterial pathogens, while leaving beneficial bacteria and human cells unharmed [ 2 , 7 – 10 ]. Phages are naturally occurring entities that infect and replicate within bacteria. They possess a high degree of specificity, with different phages targeting specific strains or species of bacteria [ 3 – 10 ]. This specificity is attributed to the recognition and binding of phage tail structures to specific receptors on the surface of bacterial cells.One of the key advantages of phages over traditional antibiotics is their ability to evolve alongside bacteria. As bacteria develop resistance mechanisms against antibiotics, phages can adapt and evolve to overcome these resistance mechanisms [ 4 – 9 ]. This dynamic nature of phages allows them to maintain their effectiveness against evolving bacterial pathogens [ 11 ] Moreover, phages have a unique mode of action compared to antibiotics. Instead of directly killing bacteria, phages replicate within the bacterial host, leading to the lysis and destruction of the bacterial cell. This lytic activity not only kills the targeted bacteria but also helps prevent the development of bacterial resistance [ 12 ]. Phages offer several other advantages as well. They have a broad range of host specificity, allowing them to target a wide variety of bacterial pathogens [ 13 ]. Additionally, phages can penetrate biofilms, which are protective structures formed by bacteria that make them highly resistant to antibiotics [ 14 , 15 ]. By effectively targeting biofilms, phages provide a potential solution to chronic and persistent infections [ 5 ]. Another benefit of phages is their relative safety. They have been extensively studied and used in certain regions for decades, particularly in Eastern Europe, as a therapeutic option for bacterial infections. Phages are generally well-tolerated by the human body and have minimal impact on the normal microbiota [ 10 – 12 ]. Phage therapy, the use of phages to treat bacterial infections, has shown promising results in both in vitro and in vivo studies. It has demonstrated efficacy against multidrug-resistant bacteria, including those that are resistant to conventional antibiotics. However, further research is needed to fully understand the potential of phage therapy and optimize its use in clinical settings. The aim of this study is to characterize the genome sequence of phage KZag1 and investigate its potential as a therapeutic agent against biofilms of K. pneumoniae .