Acinetobacter baumannii (MDR A. baumannii) is an emerging pathogen in the ESKAPE group. The global burden of antimicrobial resistance has led to renewed interest in alternative antimicrobial treatment strategies, including phage therapy. This study isolated and characterized a phage vB_AbaM_ ABPW7 (vABPW7) specific to MDR A. baumannii. Morphological analysis showed that phage vABPW7 belongs to the Myoviridae family. Genome analysis showed that the phage DNA genome consists of 148,647 bp and that the phage is a member of the Phapecoctavirus genus of the order Caudovirales. A short latent period and a large burst size indicated that phage vABPW7 was a lytic phage that could potentially be used in phage therapy. Phage vABPW7 is a high-stability phage that has high lytic activity. Phage vABPW7 could effectively reduce biofilm formation and remove preformed biofilm. The utility of phage vABPW7 was investigated in a human A549 alveolar epithelial cell culture model. Phage vABPW7 was not cytotoxic to A549 cells, and the phage could significantly reduce planktonic MDR A. baumannii and MDR A. baumannii adhesion on A549 cells without cytotoxicity. This study suggests that phage vABPW7 has the potential to be developed further as a new antimicrobial agent against MDR A. baumannii.Multidrug-resistant  MDR baumanniiA.  has become resistant to many antibiotics, including colistin, the last-resort antibiotic [baumannii in patients is associated with a variety of severe conditions such as bacteremia, pneumonia, ventilator-associated pneumonia, central nervous system infections, central line-associated bloodstream infections, catheter-associated urinary tract infections, wound infections, and surgical site infections, leading to increased morbidity and mortality, severe clinical outcomes, and longer lengths of stay at hospitals. In addition, one of the main concerns with MDR baumanniiA.  is its ability to form biofilms, the community of multiple bacterial cells associated with either biotic or abiotic surfaces. During infection by MDR baumanniiA.  in humans, MDR A. baumannii interacts and then adheres to epithelial cells. During the adherence, biofilm formation is induced, and bacterial adherence to the host cells is an essential step in MDR baumanniiA.  colonization resulting in bacterial infection and subsequent pathogenesis [A. baumannii has developed resistance to almost all of the available antibiotics [baumannii control is urgently required.Multidrug-resistant tibiotic . Infectiogenesis ,3,4. Nowibiotics . TherefoAcinetobacter phage AB02 was reported, and the phage showed therapeutic efficacy against MDR baumanniiA.  [baumanniiA.  cells and biofilm formation [Galleria mellonella [Clostridium phage phiCDHS1 reduced the bacterial attachment and cell number in human colon tumorigenic HT-29 cells [Escherichia coli (E. coli) K1 in human epithelial cells was decreased by phage K1F [Escherichia phage P1 resulted in a significant reduction in E. coli in human colon HT29 cells [baumanniiA.  in mammalian cells.Bacteriophages or phages are bacterial viruses that enter and infect bacterial cells. The replication of the phage in host cells causes bacterial lysis and death, making them an alternative antibacterial treatment ,7. Moreoaumannii . Treatmeormation ,12. Furtormation ,14, a coormation . Even thormation , Gallerillonella ,18, mousllonella , and clillonella , only a 29 cells . The inthage K1F . The tre29 cells . Howeverbaumannii.A.  The biology of the phage was characterized, and the efficacy of the phage in vitro was determined, including antibiofilm activity. To the best of our knowledge, this is the first study on the antibacterial activity of the phage against MDR baumanniiA.  in an A549 cell culture model.This study aimed to isolate a phage infecting MDR v/v) glycerol solution and were then stored at −80 °C.MDR A. baumannii , E. coli , K. pneumoniae , methicillin-resistant Staphylococcus aureus (MRSA) , and P. aeruginosa  were kindly provided by the Natural Product Research Center of Excellence, Faculty of Science, Prince of Songkla University, Thailand. A. baumannii ATCC 17978 was included. After receiving the bacterial isolates, Gram-staining and biochemical tests  were performed to confirm they were A. baumannii species. Routinely, bacterial isolates were cultured on tryptic soy agar plates . After overnight incubation at 37 °C, bacterial colonies were transferred to 3 mL of sterile tryptic soy broth . The bacterial suspension was incubated overnight at 37 °C with continuous shaking at 150 rpm. For the long-term storage of bacteria, bacterial isolates were mixed with sterile 40% (A. baumannii strains to amikacin (30 µg), ceftazidime (30 µg), gentamicin (10 µg), imipenem (10 µg), colistin (10 µg), and tigecycline (15 µg)  were tested by the disc diffusion technique. The susceptibilities of 2 in a humidified incubator.The human alveolar basal epithelial cell line A549 (ATCC Cat No. CCL-185) was provided by Professor Dr. Duncan Richard Smith, Institute of Molecular Biosciences, Mahidol University, Thailand. The cells were grown and maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% heat-inactivated fetal bovine serum  at 37 °C with 5% COA. baumannii strain ABPW063 was employed as a host for phage isolation. The wastewater samples were centrifuged to remove the debris at 6000× g, 4 °C for 10 min. The supernatant was transferred to a new tube and then filtered. Ten milliliters of the filtrate were added to 10 mL of tryptic soy broth , followed by the addition of MDR A. baumannii. The mixture was incubated at 37 °C for 24 h in a shaking incubator (150 rpm). The mixture was centrifuged to remove bacterial cells at 6000× g, 4 °C for 10 min. The supernatant was filtered through a syringe filter. A spot test was performed to detect phages. Briefly, MDR A. baumannii was mixed with top agar, and 10 µL of the filtrate was spotted onto the plate. The plates were incubated overnight at 37 °C and the lytic zone in the bacterial lawn, which indicates the presence of phages, was detected.The phage was enriched and isolated from wastewater samples collected from Thasala hospital, Nakhon Si Thammarat, Thailand. The MDR 4. 7H2O, 50 mM Tris-HCl pH 7.5). After incubation at 4 °C overnight, the samples were centrifuged at 6000× g for 10 min at 4 °C and filtered through a sterile 0.22 μm filter. The filtrate was diluted with SM buffer in a series of ten-fold dilutions followed by the double-layer agar technique. The phage was purified five times to obtain purified phage.A single plaque observed in the overlays was picked with a sterile pipette tip which was then soaked in an Eppendorf tube containing 500 μL SM buffer  and then quickly poured onto a TSA plate. Plaques were counted after incubation overnight at 37 °C.The supernatant was diluted with SM buffer by a series of ten-fold dilutions. Two hundred microliters of the phage-diluted solution was mixed with 200 µL of log-phase MDR g for 20 min at 4 °C. The supernatant was filtered through a sterile 0.22 μm filter and then concentrated using an Amicon® Ultra-15 centrifugal filter units, Ultracel 100 kDa membrane  [To amplify a phage stock, the phage supernatant was diluted with SM buffer by a series of ten-fold dilutions. The dilution (200 μL) was mixed with exponential-phase bacterial culture (200 μL) in top agar. The mixture was poured on a TSA plate and incubated at 37 °C overnight. The phage was eluted by adding 5 mL SM buffer onto semi-confluent plates. The supernatant was pooled and centrifuged to remove the bacterial cells at 6000× MA, USA) . The supv/v) uranyl acetate (pH 6.7). The phage was visualized using a JEM 2010, JEOL electron microscope  at an operating voltage of 160 kV. The phage classification based on the morphological characteristics was determined according to the standard phage classification method [A morphological observation of the phage was performed by transmission electron microscopy (TEM) as described previously . The phan method .A. baumannii, A. baumannii ATCC 17978, E. coli, K. pneumoniae, MRSA, and P. aeruginosa strains. Two hundred microliters of exponential-phase clinical bacterial isolates was mixed with top agar and poured onto a TSA plate. Ten microliters of the diluted phage stock containing 104 PFU/mL was dropped on the surface of the top agar inoculated with 200 μL of each bacterial culture. The plates were left to dry and then incubated overnight at 37 °C. The presence of a clear zone in the bacterial lawn was regarded as the lytic activity of the phage, while the absence of the clear zone indicated no lytic activity. Experiments were undertaken independently in duplicate.The standard spot test was performed to evaluate the lytic activity of the phage as previously described . The hosA. baumannii isolate. The mixtures were incubated at room temperature for 20 min, followed by the double-layer agar technique. After incubation overnight at 37 °C, the plaques were counted. The EOP value was calculated as the ratio of the titer of the phage on a test isolate to the titer of the phage on the host bacterium. Experiments were undertaken independently in duplicate.The phage lytic activity was confirmed by calculating the EOP as previously described . The spoA. baumannii was measured following a previously reported methodology [A. baumannii at an MOI of 0.1, followed by incubation at 37 °C without shaking. Samples were taken every minute for the first 10 min, after which samples were taken every 5 min from 10 to 30 min post-infection. The samples were immediately added to pre-chilled TSB supplemented with chloroform. The phage titer was then determined by double-layer agar assay. Experiments were undertaken independently in duplicate with the duplicate plaque assay. The adsorption rate constant, k, in mL/min, was calculated by following the equation as previously described [k is the adsorption rate constant (mL/min); B is the bacterial cell concentration; t is the time; Po is the stating titer; P is the final titer.The attachment rate of the phage to MDR hodology . The phaescribed .k = 2.3A. baumannii was centrifuged at 6000× g for 20 min at 4 °C. The supernatant was removed, and the bacterial pellet was re-suspended in TSB. The bacteria were infected with the phage at an MOI of 0.1. To allow for the absorption of the phage into bacterial cells, the mixture of bacteria and phage was incubated for 20 min at 37 °C. After centrifugation at 6000× g for 20 min at 4 °C to remove any unabsorbed phage, the pellet was re-suspended in TSB and then incubated at 37 °C with shaking at 150 rpm. Samples were taken every 10 min for 120 min, and the phage was titered by the soft-agar overlay method. The time between phage adsorption and cell lysis was the latent period. The burst size and the average number of phages released per bacterium was calculated from the ratio of the total number of the released phage progeny at the end of one growth cycle to the initial count of infected bacterial cells. Experiments were undertaken independently in duplicate with the duplicate assay [The latent phage period and burst size were inferred from a one-step growth curve as previously described ,27. Briete assay .A. baumannii growth was assessed following the previously established methodology [A. baumannii was cultured at 37 °C overnight, and the concentration of bacterial cells was adjusted to 1 × 108 colony-forming units per mL (CFU/mL). MDR A. baumannii was infected with phage at MOIs of 0.1, 1, and 10. The samples were incubated at 37 °C with constant shaking. The supernatant was taken every hour for 8 h to measure the bacterial growth at 600 nm. Experiments were undertaken independently in duplicate with the duplicate assay [The effect of the phage against MDR hodology . MDR A. te assay .A. baumannii cells were visualized under SEM. MDR A. baumannii cells were infected with the phage at an MOI of 1 and then incubated at 37 °C for 4 h. The samples were centrifuged at 6000× g for 10 min. The supernatant was removed, and the bacterial pellets were washed twice with 1 mL of PBS. The bacterial cells were re-suspended in 200 µL of PBS, and the bacterial suspension was dropped onto glass slides. After cell fixation with 2.5% glutaraldehyde in 0.1 M PBS, the cells were washed twice with PBS. The cells were dehydrated in a series of ethanol solutions  after which the bacterial cells were dried in a critical point dyer and then coated with gold. The ultrastructure of bacterial cells was observed under a field emission SEM (FE-SEM) .The effects of the phage on MDR 8 PFU/mL. The bacteriophage in the SM buffer was incubated at different temperatures  for 2 h. After incubation at specific temperatures, the samples were cooled in an ice-water bath, and the phage viability was determined by the double-layer agar method. The phage incubated at 4 °C was used as a control. For the ability of the phage to tolerate varying pH values, the phage was incubated in SM buffers ranging from pH 1 to pH 14 for 2 h, after which the samples at varying pH levels were neutralized to pH 7, and the phage viability was measured by the double-layer agar method. The phage incubated in the SM buffer at pH 7 was used as a control. The phage stability in glycerol stock was evaluated. The phage was mixed with glycerol in the SM buffer. The final concentrations were 25% (v/v) and 50% (v/v) glycerol [The effects of different temperatures, pH values, and UV radiation on the phage stability were measured according to the previously described methods . The phaglycerol . After iThe phage whole-genome de novo sequencing was undertaken commercially  on the Illumina sequencing platform. The phage DNA was extracted, and the quality was verified by the Picogreen method using Victor 3 fluorometry . The library was prepared using the TruSeq Nano DNA library preparation kit . After checking the library quality, the library was sequenced, and the quality of raw sequence data from high throughput sequencing pipelines was evaluated by FastQC (version 0.11.5), a quality control tool. The results from FastQC were assembled de novo into a contig by SPAdes de novo (version 3.13.0) with the k-mer parameter. Prokka (version 1.12) was used to predict the locations of protein-coding sequences, tRNA genes, tmRNA genes, and rRNA genes. BLAST and EggNOG were used to annotate the functions of genes. Blastx was performed against standard databases with an E-value cutoff of 0, a query coverage cutoff of 80%, and an identity cutoff of 80%. The genome map was constructed on the CGview server.Staphylococcus phage JD419 (QOI66744). All pairwise comparisons of the nucleotide sequences were analyzed by the Genome-BLAST Distance Phylogeny approach under settings recommended for prokaryotic viruses [The relationship of the phage to other phages in the NCBI database was determined by a Blastn search with an E-value cutoff of 0, and an identity cutoff of 70%. The outgroup was  viruses ,32,33. T viruses . Branch  viruses , and the viruses , using t viruses . The gen viruses . EMBOSS Staphylococcus phage JD419 (QOI66744) was used as an outgroup.The tail fiber gene was selected to investigate the genetic relationships using the maximum-likelihood phylogenetic tree and the Jones–Taylor–Thornton matrix-based model based on the amino acid sequence alignment. The gene was compared with other gene sequences in the NCBI database by Blastx . The mulA. baumannii was cultured and diluted with TSB. One hundred microliters of MDR A. baumannii was added into a flat-bottomed 96-well microtiter plate (107 CFU/well). Subsequently, 100 μL of diluted phage (101–108 PFU/well) was added to the wells, and the plates were incubated without agitation at 37 °C for 24 h. For the disruption of preformed biofilm, bacteria were added into a flat-bottomed 96-well microtiter plate and incubated at 37 °C for 24 h without shaking to allow for biofilm formation. At the indicated time point, the supernatant was removed. The biofilm was washed twice with PBS, and 100 µL of the phage (101–108 PFU/well) was added to the wells. The plates were incubated at 37 °C for 24 h. At the indicated time points, the plates were washed twice with PBS and then air-dried. Two hundred microliters of 0.1% crystal violet was added to stain the biofilm biomass. After incubation at room temperature for 30 min, the excess stain was washed away with PBS, and 200 μL of absolute ethanol was added to solubilize the biofilm. The biofilm biomass was measured at 595 nm using a standard microplate absorbance reader. To determine biofilm viable cell numbers, the supernatant was removed, and the biofilm was washed twice to remove planktonic bacteria with PBS. The biofilm was re-suspended in 100 µL of PBS by vigorous pipetting. The supernatant was serially diluted in PBS. Ten microliters of the supernatant were dropped onto TSA plates and air-dried. The colonies were counted after incubation at 37 °C overnight. Experiments were undertaken independently in triplicate with the duplicate assay.The ability of the phage to prevent biofilm formation and to remove biofilm was assessed according to the methods described previously . The bioA. baumannii towards A549 lung epithelial cells was examined using the MTT -2,5-diphenyltetrazolium bromide) cell proliferation assay. The cells were grown in 96-well tissue culture plates at 37 °C with 5% CO2 until the cells reached approximately 90% confluence. The medium was discarded, and cells were then washed with PBS. The cells were incubated with 200 µL of the phage (MOIs of 0.01 to 100) or MDR A. baumannii (MOIs of 0.01 to 100) diluted in cell culture medium. After incubation under standard conditions for 24 h, the medium was removed, and the cells were washed twice with PBS. One hundred microliters of complete fresh medium were added to the wells followed by the MTT solution. After incubation under standard conditions for 4 h, DMSO was added into the well to solubilize the water-insoluble formazan. Absorbance was measured at a wavelength of 570 nm using a standard microplate absorbance reader. Cells incubated with only a combination of cell culture media and SM buffer (or only cell culture media) and cells incubated with 5% DMSO were included as negative and positive controls, respectively. The experiments were undertaken independently in triplicate with the duplicate assay.The cytotoxicity of the phage and MDR The adsorption of phage to A549 cells was undertaken as described by others . BrieflyA. baumannii infection under the cell culture model was determined following the methodology in a previous report [A. baumannii at an MOI of 1. After incubation for 2 h, the cells were washed twice with PBS and then incubated with the phage at MOIs of 0.01 to 100 under standard conditions for 2 h. The medium was removed, the cells were washed twice with PBS, and complete medium was added; the cells were then cultured under standard conditions. At 24 h post-incubation, the supernatant was removed and replaced with 100 μL serum-free media containing 0.2% resazurin. The plates were incubated under standard conditions for 3 h and the fluorescence was taken at 530/590 nm to measure the reduction in bacteria. On the parallel plates, the MTT assay was undertaken as previously described. The experiments were undertaken independently in duplicate.The phage efficacy in reducing an MDR s report . The celA. baumannii were centrifuged at 6000× g for 10 min. The supernatant was removed, and the bacterial pellet was washed three times with PBS. The bacterial cells were resuspended in DMEM and then adjusted to a final OD600 of 1.0 before proceeding with the bacterial adhesion assays. After removing the cell culture medium, the A549 cells were washed with PBS and incubated with MDR A. baumannii at an MOI of 1 and phage at MOIs of 5 and 10. The cells were incubated under standard condition, and the supernatant was collected at 0, 1, 2, 4, and 6 h post-incubation. The production of planktonic bacterial cells was measured by counting the colony-forming units after plating cultures. The supernatants were serially diluted ten-fold and then spread onto TSA plates. The production of free phage was determined by counting plaques using a double-layer agar method. At 6 h post-incubation, the viability of A549 cells was assessed by a trypan blue exclusion test. The kinetics of the antibacterial activity of the phage against planktonic bacteria under the model without cells were evaluated in parallel. The experiments were undertaken independently in duplicate with duplicate plaque assay.The anti-planktonic property of phage vABWU7 under the cell culture model was assessed, and the number of planktonic bacteria and free phage production were evaluated at time intervals for 6 h. A549 cells were cultured in 6 well tissue culture plates under standard conditions and then grown until the cells reached approximately 90% confluence. Overnight cultures of MDR A. baumannii to A549 cells was evaluated as previously described [A. baumannii infection for 2 h. For the therapeutic treatment, the cells were infected with MDR A. baumannii for 2 h and then treated with the phage for 2 h. The cells were incubated under standard conditions, and the bacterial number was assessed at 0, 1, 2, 4, and 6 h post-incubation. The supernatant was discarded at the indicated time points to remove non-adherent bacteria. The cells were washed three times with PBS to remove non-adherent extracellular bacteria and phage. Subsequently, the cells were treated with 1 mL of PBS containing 0.1 mM EDTA and incubated at 37 °C for 10 min, followed by vigorous pipetting. The supernatant was serially diluted ten-fold and then spread onto TSA plates. The viable bacterial cells were determined by counting colonies formed after overnight incubation at 37 °C. In addition, the cell viability was also determined in parallel by the trypan blue exclusion test. The experiments were undertaken independently in duplicate with the duplicate assay.The effect of the phage on the adhesion of MDR escribed ,41,42,43A. baumannii that attached to A549 cells were determined following the protocol as described above, and the ultrastructure of the bacteria on the cell surface was visualized. Briefly, the phage at an MOI of 5 were added to the cells 2 h before MDR A. baumannii infection (at an MOI of 1) in prophylactic phage administration, while the cells were infected with MDR A. baumannii cells (an MOI of 1) before the phage treatment (at an MOI of 5) in the therapeutic treatment. The cells were incubated under standard condition for 4 h and then washed twice with PBS. The cells were fixed for SEM analysis following the protocol as described above. The ultrastructure of bacterial cells attached to A549 cells was observed under an FE-SEM .The effects of the phage on MDR https://www.graphpad.com/scientific-software/prism/ (accessed on 1 September 2022)) was used to analyze all data. Statistical significance analysis was undertaken by unpaired T-test using the GraphPad Prism, p < 0.05 for significance.The GraphPad Prism program, version 5 , moderate (0.5 > EOP ≥ 0.1), low (0.1 > EOP > 0.001), and no activity (EOP ≤ 0.001). Three, four, and two isolates were found to exhibit a high EOP value (EOP = 0.61–1), moderate EOP value (EOP = 0.1–0.4), and low EOP value (EOP = 0.01–0.04), respectively ) ). The phage induced blisters and pores on the bacterial cell membrane. MDR A. baumannii cells had burst, with cell lysis, leading to cell death ) c. MDR A.ll death d.v/v) and 50% (v/v) glycerol after storage at −20 and −80 °C for 30 days when compared with the stability of the phage in SM buffer at 4 °C, as a control  (Escherichia phage vB_EcoM_ESCO37 (OM386659.1), Escherichia phage phAPEC8_ev052 (LR597654.1), Escherichia phage vB_EcoM_ESCO25 (OM386656.1), Escherichia phage ESCO13 (NC_047770.1), Dompiswa phage TSP7_1 (NC_062742.1) and Klebsiella phage ZCKP1 (NC_047994.1), respectively. Staphylococcus phage JD419 (QOI66744) was used as the outgroup. The sequencing results showed that phage vABPW7 was a member of the genus Phapecoctavirus, subfamily Stephanstirmvirinae, class Caudoviricetes, and order Caudovirales.To evaluate the relationship of phage vABPW7 and other phages on a nucleotide level, the whole-genome sequence of phage vABPW7 was searched against the Blastn database and the data were further analyzed by VICTOR, a genome-based phylogeny and classification of prokaryotic viruses program. Phage vABPW7 was the most closely related to 05078.1) B. Other Escherichia phage BI-EHEC, was analyzed by ViPtree. A high identity of both genomes was observed across the majority of the genomes, although regions of lower identity were seen  and Escherichia phage vB B_EcoM_ESCO25  , a putative structural protein of Klebsiella phage ZCKP1 , a putative tail fiber protein of Enterobacteria phage ECGD1 , and a putative tail protein of Salmonella phage GEC vB MG , respectively. The tail fiber of Staphylococcus phage JD419 (QOI66744) was used as an outgroup.To analyze the relationship of phage vABPW7, a phylogenetic tree analysis was carried out with the tail fiber protein encoded by ORF159. The nucleotide sequence of the gene was searched with Blastx against the NCBI database with an E-value cutoff of 0, a query coverage cutoff of 80%, and an identity cutoff of 70%. The phylogenetic tree results showed that phage vABPW7 shared a common clade ancestor with the tail fiber protein of dentity) b. The taA. baumannii was incubated with phage vABPW7 at various concentrations. Phage vABPW7 at 101 to 108 PFU/wells were significantly effective in preventing biofilm formation by reducing approximately 9.25 to 67.27% biofilm biomass, and 91.5 to 99.4% (1.2 to 2.05 log) of viable cells compared to a control, not treated with phage ) ) ) d. Phage (n = 3)) e,f. Some(n = 3)) . The phaA. baumannii is an emerging pathogen associated with increased morbidity and mortality rates worldwide. MDR A. baumannii is frequently involved in hospital-acquired and ventilator-associated pneumonia. However, MDR A. baumannii has become a common pathogen, which can infect the respiratory tract, blood, soft tissues, and urinary tract, resulting in septicemia, meningitis, endocarditis, pneumonia, wound, and urinary tract infections. MDR A. baumannii rapidly develops resistance to all antibiotics, including colistin and tigecycline—the only effective antibiotics in clinical treatment [A. baumannii strains including the development of new antibiotics, nanoparticles, peptides, and phages. To date, various phages infecting A. baumannii have been studied and reported, and Acinetobacter phages have been investigated in vitro and in vivo, including their use in clinical trials in the US [Acinetobacter phage in a cell culture model. To further shed some light on phage therapy, this study focused on the isolation and characterization of a phage infecting MDR A. baumannii, determined the effects of this phage on biofilm, and evaluated the efficacy of phage in vitro, including in an A549 cell culture model.MDR reatment . Given tn the US ,48. HoweA. baumannii lawn, and the plaques have a halo around them indicating that the phage might encode depolymerases with polysaccharide-degrading activity. A phage host range and EOP analysis showed that phage vABPW7 was able to infect 45% of 20 clinical strains. These results indicated that phage vABPW7 could possibly be used in phage therapy, especially as part of a phage cocktail. An increase in the number of bacterial strains for host range testing might provide useful additional information. Many common factors affecting phage viability, such as temperature, pH, UV radiation, and phage infectivity over long periods, have been reported [A. baumannii completely, highlighting the high lytic activity of phage vABPW7.Phage vABPW7, which has a long and contractile tail, was enriched and isolated from a wastewater sample. The phage produces small plaques on an MDR reported . Phage vPhapecoctavirus, subfamily Stephanstirmvirinae, class Caudoviricetes, and order Caudovirales. The comparative whole-genome sequencing results showed no Acinetobacter phage-sharing whole-genome similarity with phage vABPW7. Interestingly, phage vABPW7 shares the highest whole-genome identity with Escherichia phage BI-EHEC. The phage also shares high sequence similarity with Klebsiella phage ZCKP1, Dompiswa phage TSP7_1, and Myoviridae sp. Even though most of the studies on phages have reported that phages are highly host-specific, some phages can infect many species and different genera of bacteria [Escherichia phage, could infect A. baumannii. The identity of phage vABPW7 and Escherichia phage BI-EHEC was 95.1%, indicating that phage vABPW7 might be a typical phage in the genus Phapecoctavirus, subfamily Stephanstirmvirinae, class Caudoviricetes, and order Caudovirales. Furthermore, there was no virulence factor or resistance gene in the genome of phage vABPW7, suggesting the potential as a reliable therapeutic agent for phage therapy against MDR A. baumannii. Due to the high similarity of phage vABPW7 and Escherichia phage-specific avian pathogenic E. coli, the lytic activity of phage vABPW7 towards Avian pathogenic E. coli should be investigated in the future.The genome study of phage vABPW7 showed that the phage is classified as a member of the genus bacteria . Thus, iEscherichia phage vB B_EcoM_ESCO25 and Escherichia phage ESCO13, demonstrating that phage vABPW7 was grouped into the genus Phapecoctavirus, corresponding to the whole-genome analysis. In addition, previous studies have shown that the tail fiber proteins of phage, such as Acinetobacter phage IME200 [Acinetobacter phage phiAB6 [Klebsiella phage SH-KP152226 [The process of a phage entering bacterial cells is mediated by a specific interaction between the bacterial cell membrane and the tail fiber protein, which is a phage receptor-binding protein functioning in phage adsorption and penetration into bacterial cells . The bine IME200 , Acinetoe phiAB6 , and KleKP152226 , possessKP152226 . CapsulaKP152226 . It is wKP152226 , and as KP152226 . MoreoveA. baumannii can cause various diseases, including pneumonia, an infection that inflames the alveoli in the lung. Cases of nosocomial pneumonia, severe community-acquired pneumonia, and ventilator-associated pneumonia have been reported to continuously increase [A. baumannii can adhere, colonize, and invade human epithelial cells, followed by intracellular multiplication, dissemination to other tissues, persistence, and activation of cell death pathways [A. baumannii infections in mammalian cells. A. baumannii interacts with A549 cells through fibronectin and then enters the cells, leading to pathogenesis [MDR increase . Previoupathways . Human logenesis ,63. Receogenesis . In addiogenesis . To ensuogenesis . Moreoveogenesis . In an aogenesis . Howeverogenesis , and theAcinetobacter phage vABPW7 has the potential to be used as an alternative antibacterial approach to control MDR A. baumannii infections. Phage vABPW7 is suitable for further development towards its application in phage therapy, including as part of a phage cocktail.


Bacteriophage (phage) therapy is re‐emerging a century after it began.Activity against antibiotic‐resistant pathogens and a lack of serious side effects make phage therapy an attractive treatment option in refractory bacterial infections.Phages are highly specific for their bacterial targets, but the relationship between in vitro activity and in vivo efficacy remains to be rigorously evaluated.Pharmacokinetic and pharmacodynamic principles of phage therapy are generally based on the classic predator–prey relationship, but numerous other factors contribute to phage clearance and optimal dosing strategies remain unclear.Combinations of fully characterised, exclusively lytic phages prepared under good manufacturing practice are limited in their availability.Safety has been demonstrated but randomised controlled trials are needed to evaluate efficacy. Shigella enteritis (bacillary dysentery) in 1919, in the first recorded description of phage therapy. In the years that followed, phages were used with varying success by d'Herelle and others for a variety of serious infections, including staphylococcal bacteraemia, typhoid fever and osteomyelitis,Caudovirales)  . Phages Box 1A) and electron micrograph of bacteriophage (Myoviridae) with 1% uranyl acetate negative staining, with size marker (B). Bacteriophage preparations were dialysed against 0.1 M ammonium acetate in dialysis cassettes with a 10 000 membrane molecular weight cut‐off (Pierce Biotechnology), negatively stained with 2% uranyl acetate, and visualised using transmission electron microscopy (TEM). TEM was conducted at the Westmead Scientific Platforms  on a Philips CM120 BioTWIN (Thermo Fisher Scientific) transmission electron microscope at 100 kV. Images were recorded with a SIS Morada digital camera using iTEM software (Olympus Soft Imaging Solutions).Schematic  in the 1970s of high dose phage for the treatment of cholera found tetracyclines to be more effective.Escherichia coli infection, and only half of these were phage‐susceptible in vitro. More recently, a phase 1 double‐blind RCT of a topical bacteriophage cocktail in chronic venous leg ulcers found no safety concerns,Pseudomonas with standard care with silver sulfadiazine, found that significant reductions in bacterial counts took longer in the phage group and that the silver sulfadiazine group had higher treatment success than the phage group. The PhagoBurn trial suffered from instability of the phage preparation, but showed that phage‐susceptibility of Pseudomonas isolates in vitro is crucial to eventual clinical success, with susceptibility rates of 89% in successful cases compared with 24% in clinical failures in the phage therapy arm  experience in humans is less impressive . The larrapy arm .Box 2Phages usually exhibit a high degree of host specificity and are most easily sourced from the habitat of their usual bacterial hosts. Phages specific for clinically relevant bacteria may be readily sourced from human and animal sources, hospital wastewater, and environmental soil and water. Their reproduction is dependent on the bacterial host, either integrated into the bacterial genome as a prophage or acting in a purely parasitic manner , the latBox 3A), the phage DNA (purple) is classically either reproduced and packaged as new virions at the expense of the cell  or reproduces with the host DNA .After infection  susceptible isolate — productive infection and formation of individual plaques detectable at serially diluted phage lysate — and (B) resistant isolate — single plaques absent and early bacterial lysis present only at high concertation of phages. Liquid culture‐based system: (C) across a gradient of multiplicity of infections (MOIs) against eight bacteria on a single 96‐well plate. There are seven MOIs tested from highest to lowest (left to right). Column eight and nine are phage‐only and bacteria‐only controls. (D) Bacterial growth kinetics in presence of phage at differing MOIs.Phage‐susceptibility testing of bacterial isolates with the standard double‐layer method:The plaque assay is time‐consuming and operator‐dependent, with automated methods still in their infancy. Liquid culture‐based systems that continuously monitor bacterial growth in the presence of bacteriophage in a standard 96‐well plate format , C may bAcinetobacter infection when antibiotics were failing.The development of in vitro bacterial resistance was a prominent feature of phage therapy for a high burden multidrug‐resistant input)A good understanding of the non‐linear kinetics of bacteriophage distribution in blood and tissues is necessary to maximise efficacy of future therapeutic protocols.1–105  colony‐forming units per mL of blood in severe sepsis,9 plaque‐forming units (PFU) into the human blood volume (~ 5 L) is expected to yield an MOIinput over 200.Numerous methods of bacteriophage delivery have been explored, including topical, inhalational, oral and injectable . When administering phages orally, concern has been raised regarding recombination between modular phage genomes in the gut, although there has been little evidence from trials to date to resolve this one way or the other.The paradoxical persistence of a narrow host range for phages is not fully understood, given the presumed ecological advantagesE. coli have added additional barriers (curli fibres) as a collective protection.Urine concentration of bacteriophage may be dose‐dependant,Optimised preparation of phage combinations (cocktails) for use in humans requires well characterised bacteriophages, a good understanding of the bacterial target population, and highly effective purification protocols to avoid inflammatory responses to contaminating residual bacterial endotoxin and protein.9–1010 PFU/mL) with unique but overlapping host ranges to guarantee lysis of the bacterial target and clinically relevant variants.General recommendations regarding the standardisation of key components of phage therapy have been published,Phage therapy may be presented as a discrete quality‐assured formulation either as several phages admixed to provide a broad spectrum of activity in a single medicine for a specific infection  clinical trials in humans means that phage therapy still has the status of an old technology based on anecdotal evidence, not much different to traditional medicines. Nevertheless, there are some successful phase 1 and 2 studies, including in Australia, which suggest the potential of phage therapy as an alternative or adjunct to antibiotics.S. aureus sepsis and infective endocarditis were treated twice daily for 2 weeks with intravenous AB‐SA01  — a GMP‐quality cocktail product with three bacteriophages — as adjunct to standard care.Recent studies in Australian centres of both intranasal instillationNatural bacteriophages are defined as investigational drugs by the Therapeutic Goods Administration in Australia. The regulatory side of bacteriophage therapy is beyond the scope of this review, but most medical communities are both sceptical and curious.To progress, we need at least to guarantee the availability of efficient phage susceptibility testing and of preparations that are safe for intravenous administration, define optimal dosing, and effectively monitor phage–bacteria–human host interactions and any important collateral effects on other members of the microflora.Realistically, application of therapeutic phages alone may never completely replace chemical antimicrobials as the standard of care. For now, it is reasonable to define phage therapy as a promising rescue therapy that has been associated with some spectacular resultsNo relevant disclosures.Not commissioned; externally peer reviewed.


Escherichia phage, YF01, from a municipal wastewater treatment plant in Yokohama, Japan. We demonstrate that the YF01 phage shares a high similarity to a collection of thirty-five Escherichia and Shigella phages found in public databases, six of which have been previously classified into the Kuravirus genus by the International Committee on Taxonomy of Viruses (ICTV). Using modern phylogenetic approaches, we demonstrate that an expansion and reshaping of the current six-membered Kuravirus genus is required to accommodate all thirty-six member phages. Ultimately, we propose the creation of three additional genera, Vellorevirus, Jinjuvirus, and Yesanvirus, which will allow a more organized approach to the addition of future Kuravirus-like phages.Bacteriophages, viruses that infect bacteria, are currently receiving significant attention amid an ever-growing global antibiotic resistance crisis. In tandem, a surge in the availability and affordability of next-generation and third-generation sequencing technologies has driven the deposition of a wealth of phage sequence data. Here, we have isolated a novel  Escherichia coli is a versatile and genetically diverse species commonly inhabiting the intestinal tracts of humans and animals as both a commensal and pathogenic microbe [E. coli infecting humans are routinely released into the environment via sewage systems, which lead to wastewater treatment plants (WWTPs) [E. coli, alternative methods to control these pathogens are highly sought after. Bacteriophages (or phages) have received high attention as a potential therapeutic alternative to antibiotics [ microbe . Pathoge (WWTPs) . Given tibiotics ,4,5. 8–109 particles per milliliter [E. coli in biological WWTPs [Phages are viruses that multiply by infecting bacteria, often very specifically at the species or strain level. They play a key role both in microbial ecology and microbial evolution, having the ability to alter the population dynamics within microbial communities and modify bacterial genomes through horizontal gene transfer ,6,7. In lliliter , which ial WWTPs . The useal WWTPs . The advent of next-generation sequencing technologies, such as those offered by Illumina, has led to a preponderance of genomic sequencing. Newer “third-generation sequencing” technologies, such as those offered by Oxford Nanopore, facilitate the acquisition of long-sequence reads (up to Mbs), allowing for the generation of complete bacterial and other higher organism genomes. While commonly used in combination with highly accurate short-reads, more recent revisions of the technology, known as Q20, include qualitative improvements that facilitate the generation of near-perfect bacterial assemblies, and perfect viral assemblies, without the need for short-read polishing ,15.Escherichia phage assemblies listed on NCBI Genbank. The International Committee on Taxonomy of Viruses (ICTV) currently recognizes Escherichia phages belonging to at least 11 different families and 100 different genera [Phieco32-like virus genus was created in 2009, in response to the isolation and characterization of the Escherichia phage phiEco32, a novel 77 kb phage with a rare C3 podovirus morphotype [Phieco32likevirus and then Phieco32virus), while also adding five new phage members to its ranks  [Kuravirus in 2019.As of December 2022, there are currently ~1600 rphotype . The gen kv1721) ,19,20. TE. coli phage, YF01, from a WWTP in Yokohama, Japan. The YF01 phage was sequenced and assembled using long-read Q20 Oxford Nanopore technology and shown to be related to phages from the ICTV-classified Kuravirus genus. Modern reticulate network analyses revealed that YF01 and the current six ICTV-classified Kuravirus phages shared high similarity with 29 other phages present in public databases. Using a combination of approaches, we demonstrate that these 36 phages represent four different genera under current ICTV guidelines. We further analyze the genomic features of the YF01 phage in comparison to other Kuravirus-like group phages and elucidate the core proteome of the group to demonstrate that the large terminase subunit and portal protein, among three other proteins, show high conservation and highly correlated phylogenetic reconstruction to whole genome-based methods. Ultimately, we recommend the expansion and reshaping of the Kuravirus genus to four separate genera to accommodate a total of 36 phages isolated worldwide.In this study, we isolated a novel Escherichia coli strain K12 (JCM20135), isolated from feces from a diphtheria convalescent sample, was used in this study. Bacterial cultures were grown in Nutrient Broth with NaCl  and NB with agar (with added 12 g L−1 agar) at 25 °C. Cultures were grown under aerobic conditions.g for 5 min. The supernatant was filtered through a cellulose acetate (CA) filter with a 0.20 μm pore size . A total of 1 mL of filtrate was added to NB and 100 µL of log phase E. coli K12. The suspension was incubated overnight at 25 °C. The overnight culture was centrifuged at 2000× g for 10 min and the supernatant filtered through a CA 0.20 μm pore filter . E. coli colonies from a growth plate were taken with sterile cotton swab and spread uniformly on NB agar medium. A total of 40 μL of filtrate was added plate dropwise, air-dried, and incubated at 25 °C overnight. A visible plaque was removed with the wide end of a tip and resuspended in SM buffer , a process repeated twice to ensure a pure phage isolate. Activated sludge mixed liquor was collected from a municipal WWTP in Yokohama in November 2021 where urban wastewater was received. The activated sludge sample was transferred to a 15 mL tube and centrifuged at 2000× 11 PFU mL−1) was incubated on a grid for 10 min. Grids were washed twice in MilliQ water and stained with 2% (w/v) uranyl acetate prior to a final wash in MilliQ water. Grids were dried prior to examination under a JEOL JEM02010HC electron microscope. TEM was performed as previously described . Briefly11 PFU mL−1) were mixed in a 9:1 ratio and incubated at 25 °C for 1 h. Phage PFU mL−1 was then determined by spot test (20 µL) using a dilution series down to 10−8 and counting the dilution that resulted in 10–100 plaques. For temperature testing, the NB and YF01 phages (~1 × 1011 PFU mL−1) were mixed in a 9:1 ratio and incubated for 1 h at different temperatures . Phage PFU mL−1 was determined as above. Each experiment was repeated two times.To test phage stability, the pH of NB was adjusted to 3, 5, 7, 9, and 11 using NaOH and HCl. The pH-adjusted NB and YF01 phages (~1 × 1010 PFU mL−1) was extracted using a zinc chloride phenol:chloroform extraction method [2 to remove host contaminants before the precipitation of phage virions by the addition of 40 mM ZnCl2. Virions were resuspended in phage extraction buffer  SDS and 50 µg mL−1 proteinase K) and incubated for 1 h at 55 °C. An equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) was added and the top layer was removed and DNA precipitated with isopropanol. The pellet was washed in 70% (v/v) ethanol and resuspended in 10 mM Tris-HCl (pH 8.5). DNA concentration and purity were measured using a Quantus Fluorometer  and NanoDrop One , respectively. DNA integrity was assessed via agarose gel electrophoresis.Genomic DNA from 1 mL phage filtrate (>1 × 10n method . Brieflyhttps://github.com/rrwick/Filtlong). Filtered mean read size was 17.8 kb with a mean read quality of Q20.3, as assessed by NanoPlot v1.40.0 [https://github.com/nanoporetech/medaka) made no corrections to the assembly. Residual Nanopore adapter sequences were truncated from the termini of the final assembly by screening against Y-adapter top (5′-GGCGTCTGCTTGGGTGTTTAACCTTTTTTTTTTAATGTACTTCGTTCAGTTACGTATTGCT-3′) and Y-adapter bottom (5′- GCAATACGTAACTGAACGAAGT -3′) sequences.DNA libraries were prepared using 1 ug of DNA using the Ligation Sequencing Kit 14 (SQK-LSK114), loaded on a R10.4.1 flow cell (FLO-MIN114) and sequenced using a MinION Mk1B . Simplex and duplex basecalling was performed using Guppy v6.4.2 in super accurate (SUP) mode. Read filtering was performed with Filtlong v0.2.1 . DTR sequences in other eviously . To annoeviously  and manueviously , a profieviously , and theeviously . tRNAs weviously  and Arageviously . Figureseviously . Amino aeviously  in GeneiE. coli K12 MG1655 (NC_000913). Codon usage was determined using the statistics function in Geneious Prime v2022.2.1. Codon usage that differed by ≥2-fold compared to the host were plotted using Prism v7.0.Coding sequences (nucleotide) for YF01, phiEco32, ES17, KBNP1711, and ECBP2 phages were determined using Prokka and directly accessed from Genbank for Kuravirus-like group phages was performed using Roary [The core analysis of the ng Roary . Brieflyng Roary  was usedng Roary  and the Escherichia phage YF01 was isolated from activated sludge collected from a municipal WWTP in Yokohama, Japan. The phage formed small, transparent, circular plaques approximately 1 mm in diameter (−1) noted from 60 °C and no observable phage activity above 70 °C (diameter a. Phage diameter b. We nexve 70 °C c. The YFve 70 °C d. The pHE. coli (~50.8%), a common phenomenon among lytic phages [The YF01 phage was sequenced using long-read Q20 Oxford Nanopore technology, leading to the assembly of a 78,626 bp genome with 704-fold coverage . The GC c phages .Escherichia (and 1 Shigella) phages  . As vCongrouping ) of 24 aKuravirus phages, and performed matrix nucleotide identity analysis to understand the nucleotide-level similarity more precisely. Using nucleotide similarity thresholds of 95% and 70%, for species and genus demarcation, respectively, as recommended by the ICTV, VIRIDIC grouped these phages into a total of thirty-three species and four genera G1-G4  grouped into the largest genus (G1), while KBNP1711 and ECBP2 phages grouped into two separate genera, G3 and G4, respectively. The apparent discrepancy between our genera groupings and the Kuravirus classifications by ICTV occurred due to the nucleotide similarity between the KBNP1711 and ECBP2 phages and the other ICTV-classified phages (such as the phiEco32 phage) falling below the currently accepted threshold of 70% .We took these 36 grouped phages, including the YF01 phage and the six ICTV-classified ra G1-G4 . In contAeromonas, Cronobacter, Escherichia, Proteus, Salmonella, and Vibrio species. VICTOR phylogeny demonstrated that the 36 clustered phages formed a monophyletic group , using whole phage genome sequence inputs . Here weic group , with clic group , G1–4. OKuravirus phages occurred between 2009 (the creation of the genus with phiEco32 phage) and 2015–2016 . Given the substantial increase in the amount of Kuravirus-like phage genomes deposited in public databases since then (30 new Kuravirus-like phages deposited in the past 9 years) and based upon the current criteria for the genome-based classification of viruses [Kuravirus genus into four separate genera based on the currently accepted similarity thresholds . We will recommend to the ICTV the identification of these four genera as Kuravirus (G1), Vellorevirus (G2), Jinjuvirus (G3), and Yesanvirus (G4), with the names of the new genera (G2–G4) representing either the geographical location of isolation or locality of the research group, of the founding phage member of each respective genus . At the present time we will refer to this grouping of 36 phages as the Kuravirus-like phage group or simply “the group”  and genes involved in DNA replication and nucleotide metabolism on the right  direct terminal repeats (DTRs) indicative of a T7 phage-like replication strategy where the substrate for capsid packaging are large DNA concatemers. The presence of DTRs in the group was first determined in phiEco32 phage (193-bp DTRs) using a combination of restriction profiling, primer walking, and sub-cloning strategies [rategies . In morerategies . With thrategies ,61. Givegp3-4), which form the molecular motor responsible for packaging of the nascent phage DNA, are the first two genes in this module. While gp3 displayed no known domains via the NCBI conserved domains database (CDD), it did show similarity to the small terminase subunit in the Pseudomonas phage PaP3 using a more sensitive comparison of profile hidden Markov models (HMM) [gp25 in the YF01 phage) annotated as a terminase small subunit. This was likely due to the presence of a HNH endonuclease domain, which are often associated with phage DNA packaging [gp3 as the putative small terminase subunit in the YF01 phage. The virion morphogenesis and lysis module in the YF01 phage shares high homology with other group phages, comprising a total of 20 genes transcribed in the forward direction . The terls (HMM) ,64. We dackaging . Howevergp5-9) encode proteins that assemble to form the rare C3 morphotype (an elongated phage capsid) seen in the YF01 phage (gp5), scaffolding protein (gp7), major capsid protein (gp8), and adapter protein  and a larger product that occurs due to a ribosomal slippage event at a heptanucleotide slippery sequence  near the natural stop codon of gp8, leading to an extended isoform using the -1 frame . This is a well-known phenomenon in the tail assembly chaperone in long-tailed phages (~3.5% slippage rate in the Escherichia phage Lambda [Escherichia phages T3 and T7 (~10% slippage rate) [The next genes in this module  but alsge rate) ,68. Kuravirus-like group phages remains unclear. In the YF01 phage, the extended MCP isoform is substantially larger (892 a.a) than those seen in many other group members (~500–550 a.a) and most similar in size to that seen in the Paul phage (894 a.a.) . As obseInterestingly, the ultrastructural analysis of the group member, SU10 phage, revealed a capsid structure with apparent uniform MCP formation  . Consistgp12) and an endolysin (gp13), which work in concert to induce host cell lysis at the conclusion of the phage lifecycle  and distal (gp11) tail fibers likely assemble into hexameric long tail fibers, which are attached to the adapter complex [gp15), also attached to the adapter complex, is bound by short tail fibers (gp14) [gp16, extends from the channel formed by the nozzle.The next structural genes in this module encode numerous tail proteins ( complex . The nozs (gp14) . The thrs (gp14) , and maygp18-22) and may be packaged inside the phage capsid along with the genomic DNA and play roles during the infection process gp49) and separately encoded 5′-3′ exonuclease (gp27) and 3′-5′ exonuclease (gp70) subunits. The separation of the DNA polymerase and its exonuclease domains is common to all group members and is similarly observed in phages closely related to the group  and NAD-dependent DNA ligase (gp57). The YF01 phage, like many phages, encodes certain enzymes involved in nucleotide metabolism and modification. These include a deoxynucleoside monophosphate kinase (gp28), a thymidylate synthase (gp60), and a deoxycytidine triphosphate deaminase (gp68).The genome of the YF01 phage contains an assortment of genes involved in DNA replication, repair, and nucleotide metabolism. These include a DNA polymerase (gp30) and a small transcriptional regular (gp76), which are highly conserved across the phage , middle genes located in the middle of the phage genome corresponding to DNA replication genes (gp23-81 in the YF01 phage), and late genes located at the left of the phage genome corresponding to the virion morphogenesis and lysis genes (gp1-22 in the YF01 phage). In the phiEco32 phage, RNA polymerase holoenzymes assembled with the phage-encoded sigma factor (gp30 in the YF01 phage) were shown to drive expression of some middle genes and all late genes  [gp76 in the YF01 phage) appeared to have a dual function in the phiEco32 phage by (1) promoting the expression of the phage sigma factor and (2) shutting off early gene expression via physical interaction with host σ70-factor, essentially promoting the progression of the phage lifecycle to late stage genes [Within the YF01 phage genome, we also identified genes encoding an RNA polymerase sigma factor (n cycle) . The trage genes .E. coli strain (K12), AGA was the second least used of five possible Arg codons, with a usage share of approximately 3.6%. This represents a ~5.6 fold increase in AGA codon usage in the YF01 phage over the host  revealed a core proteome of 63 proteins, which were shared amongst all member phages (53% of the total proteome in the case of YF01 phage; Finally, we wanted to determine the core protein-encoding genome of the Kuravirus genus from six to thirty-six member phages using a combination of modern bioinformatics approaches—here grouped into four genera, namely Kuravirus and the three newly proposed genera, Vellorevirus, Jinjuvirus, and Yesanvirus. We also described a new member of this group, the Escherichia phage YF01, isolated from activated sludge in Yokohama, Japan. Genomic analyses described the organization of YF01 phage and characterized the function of encoded proteins involved in phage DNA replication and metabolism, virion morphogenesis, host cell lysis, and transcriptional regulation. Lastly, we determined that sixty-three proteins form the core proteome of Kuravirus-like group phages, with five proteins, including the large terminase subunit and DNA primase/helicase, as some of the strongest phylogenetic markers of Kuravirus-like phages.This study describes the expansion and reshaping of the members from the current ICTV-classified


Bacteriophages (phages) are selective viral predators of bacteria. Abundant and ubiquitous in nature, phages can be used to treat bacterial infections (phage therapy), including refractory infections and those resistant to antibiotics. However, despite an abundance of anecdotal evidence of efficacy, significant hurdles remain before routine implementation of phage therapy into medical practice, including a dearth of robust clinical trial data. Phage–bacterium interactions are complex and diverse, characterized by co‐evolution trajectories that are significantly influenced by the environments in which they occur . An understanding of the molecular mechanisms underpinning these dynamics is essential for successful clinical translation. This review aims to cover key aspects of bacterium–phage interactions that affect bacterial killing by describing the most relevant published literature and detailing the current knowledge gaps most likely to influence therapeutic success. This comprehensive review discusses bacterium–phage interactions, antimicrobial potential of phages and research challenges that impact realization of the successful bacteriophage therapy. BacteriophagesBacteriophages, or phages, are viruses that specifically and selectively infect bacteriaBiofilmSurface‐attached, structured community of microorganisms embedded in a self‐produced extracellular matrix EnzybioticsPhage‐derived antibacterial enzymes with therapeutic potential. Depolymerases catalyse the hydrolysis of the capsule polysaccharide of Gram‐negative bacteria, while lysins  are hydrolytic enzymes capable of cleaving the cell wall (peptidoglycan) of both Gram‐negative and Gram‐positive speciesL‐formsCell wall‐deficient bacteria resistant to supra‐therapeutic concentrations of cell wall targeting antibioticsLysogenic conversionPhage–bacterium interaction in which a prophage encodes proteins that enhance bacterial fitness or virulenceLysogeny or lysogenic cyclePhage life cycle in which the viral genome stably integrates in the bacterial chromosome, replicating with itLytic infection and productive lysisInfecting phages replicate their genome and assemble new viral particles (virions) by hijacking host resources. Phage‐directed cell lysis then releases this viral progeny ready to infect new cells, in an exponential growth cycle (productive lysis) limited only by availability of bacterial prey and their response/s to phage attackObligate lytic phagesPhages that cannot undergo lysogeny. Preferred for therapeutic applicationsPhage adsorptionMolecular interactions between phage proteins and specific bacterial receptors that bind the phage to the bacterial cell surface allowing for infection (phage genome release into the cytosol) to occurPhage cocktailCombination of multiple phages for therapeutic application. Phages in a cocktail ideally act synergistically against a bacterial target and limit the development of phage‐resistant variants. Cocktails combining phages with different host specificity allow for broader therapeutic targetingPhage therapyMedical use of phages as antimicrobials for treatment of bacterial infectionsPseudolysogenyPhage–bacterium interaction in which the phage genome resides within the host cell without chromosomal integration, in an unstable, inactive stateTemperate phagesPhages capable of undergoing lysogeny. These may lie “dormant” within a living bacterial cell while integrated into the host chromosome as “prophages”, but have the potential to enter a lytic infection cycle under certain conditions (e.g. host cell stress). Temperate phages are less preferred for therapyTransductionPhage‐mediated horizontal transmission of genetic information from one bacterial cell to another, as opposed to genetic inheritance through reproduction . Mainly associated with the lysogenic life cycleth century saw an unprecedented steady decline in mortality attributable to bacterial infections ], and immediately recognized their potential as antimicrobial agents  of antibiotics has since led to the emergence of globally disseminated bacterial pathogens that are resistant to last‐line treatments, and antibiotic resistance now poses a significant global health and economic burden , phages have been in routine medical practice for over 70 years and this experience provides a rich source of empirical data  constitute the largest group described to date  within minutes of infection  is pragmatically addressed by the use of combinations of multiple phages (cocktails) with differing adaptive strategies . StableInoviridae    Fig   and the absence of classic lysogeny genes , but there is no established genetic marker of pseudolysogenic capacity, as it is not usually a feature of exponentially growing bacteria. Replication of obligate lytic T4‐like phages is completely inhibited in nutrient‐stressed E. coli, but it has been reported that under the same conditions, a T4rI mutant (defective in the function of the holin inhibitor) keeps producing viable virions , fully resuming in the rest of the population only upon nutrient addition with restoration of logarithmic growth. P. aeruginosa and Yersinia enterocolitica can support pseudolysogenic infection with apparently obligate lytic myoviruses or podoviruses that provide bacteria with immunity from further phage infection (superinfection exclusion)  physical protection from harsh environmental conditions outside the host   antibiotics with important clinical implications  and host control of the administered therapeutic virus by innate and acquired immune responses  is the term used to indicate the ratio of phages to bacteria in esponses . The phas et al, . Carefuls et al, .et al, et al, Phage adsorption to the bacterial cell is a first and crucial step in the infection process  also play a unique role in controlling phages as they can effectively trap them, preventing host infection   actually able to succeed in nature , resulting in host range expansion , phage type (even very closely related phages can give different outcomes), and timing of administration  , the testing conditions , l, et al  presenteet al, et al, in vivo can be problematic . Given the ubiquity of phages in nature and the aeons of co‐evolution with bacteria, an array of finely tuned and well‐established defences against phage attack are also to be expected. The physiological state of the bacterial host population is an important determinant of phage replication , and theet al, et al, et al, et al, et al, The impact of bacterial stress on the lytic/pseudolysogenic pathways may be therapeutically important. Phages that ordinarily pseudolysogenize stressed bacteria , both Gram‐positive and Gram‐negative bacteria live in complex sessile biofilm communities  phage preparations may induce the expression of key anti‐inflammatory genes, including IL‐1RA and IL‐10 family cytokines  in T4‐like phages , which can impact phage bioavailability Phages that have evolved to protect their prey populations by down‐regulating the host immune response may prove to be difficult choices in therapy. Conversely, phage‐mediated immunomodulation may be a good therapeutic trade‐off in severe sepsis where attenuation of a lethal cytokine‐mediated inflammatory response may be the most important therapeutic goal.In this review, we sought to highlight the main areas of phage and bacterial biology that may directly relate to therapeutic outcome and in need of further investigation Table .et al, However, bringing phages into the pharmacopoeia requires attention to several other areas that we have not fully discussed. The limited host range of most therapeutic phages means that this precision therapy needs well‐curated and accessible phage sources, which is a biobanking and information management challenge  —https://phage.directory/Phage Directory —P.H.A.G.E. vzw ‐ Home (p‐h‐a‐g‐e.org)Phages for Human Applications Group Europe —https://phageaustralia.org/Phage Australia —http://www.yalephagecenter.com/Center for Phage Biology and Therapy at Yale —Center for Innovative Phage Applications and Therapeutics (ucsd.edu)Centre on Innovative Phage Applications and Therapeutics (first dedicated phage therapy centre in North America)—Online links to relevant sources


Validated methods for phage selection, host range expansion, and lytic activity determination are indispensable for maximizing phage therapy outcomes. In this review, we describe some relevant methods, highlighting their advantages and disadvantages, and categorize them as preliminary or confirmatory methods where appropriate. Experimental conditions, such as the composition and consistency of culture media, have an impact on bacterial growth and, consequently, phage propagation and the selection of phage-resistant mutants. The phages require different experimental conditions to be tested to fully reveal their characteristics and phage therapy potential in view of their future use in therapy. Phage lytic activity or virulence should be considered as a result of the phage, its host, and intracellular/environmental factors, including the ability of a phage to recognize receptors on the bacterial cell surface. In vitro quantitative and qualitative measurements of phage characteristics, further validated by in vivo experiments, could be incorporated into one system or mathematical model/formula, which could predict a potential successful outcome of clinical applications. A century ago, bacteriophages (phages) were defined as “devourers of bacteria”  or “obliPhages are the most abundant and diverse life-like entities on Earth, where they are found in almost all ecospheres, such as seas, rivers, and soil, and within other organisms, including humans. They control the abundance of their bacterial hosts and, as such, also impact global energy and nutrient cycles . Phages virulentus, meaning “full of poison”. They are used to indicate the relative capacity of a “microbe”  to cause disease [The words “virulence” and “virulent” come from the Latin word  disease , or in c disease ,13. Lyti disease . Gill et disease . Phage v disease , and may disease . Sometim disease .In fact, virulence is not a distinct phage characteristic, but a complex, dynamic, and variable phenomenon that includes both phage and bacterial factors . Indeed,Pseudomonas aeruginosa than either one acting alone [Nowadays, experts increasingly agree that phages will not replace antibiotics , and coung alone . Phages ng alone  one of tng alone , and preng alone , are of ng alone ,26,27 toMycobacterium smegmatis “surrogate” strain for the propagation of M. tuberculosis phages [Today’s laboratory facilities and materials are more developed than those in Félix d’Hérelle’s time. Glass tubes and Pasteur pipettes, for instance, are replaced with Eppendorf tubes or 96-well microtiter plates and multichannel micropipettes. Notwithstanding the modernization of laboratory equipment, there are no significant differences in the techniques used for phage isolation and propagation, the development of phage cocktails, nor the  production of therapeutic phage preparations. In 1930, d’Hérelle recognized that the most effective therapeutic phages could be isolated from patients that had recovered from infection. He also claimed that more than 50 bacterial strains should be used in phage isolation and enrichment methods . Interess phages  to impros phages . Notwiths phages ,32 to des phages  or perfos phages , for inss phages .This review brings together relevant methods for phage isolation, detection, characterization, and selection, including phage activity determination, host range evaluation and expansion, and the translation of in vitro results to clinical practice. We will mostly focus on the practical side of these methods , including some inputs and interpretations based on our personal experiences, as well as the advantages and disadvantages of the methods with regard to developing more standardized approaches.In this section, we will discuss a number of methods that are commonly used for in vitro phage lytic activity determination, including phage detection and enumeration testing and the in vivo translation of results (Diagram 1).Bacteria hooks with hosts covering the wide range of receptors needed to hook the largest variety of potential phages. This requires having a readily available panel of strains with known genetic profiles. Every newly isolated phage can be further studied, e.g., to determine its biology;Bacteria hooks of particular interest can be included. In this case, bacterial strains are selected based on specific features such as antibiotic resistance, and it is not necessary to have an exhaustive list of characteristics or to know their genomic profile. The strains could be objects of further scientific study.Bacterialstrains used for the “fishing” or detection of new phages are referred to here as “bacteria hooks”. For the isolation of potentially new phages, the well-known “phage enrichment” (PE) method is used. It was first developed by Winogradsky and Beijerinck  and lateTwo times  or ten tIt is preferable not to centrifuge/filter the sampling source, unless it contains large contaminants and/or components that will interfere with the incubation process. It is assumed that conditions close to those in the natural source environment will facilitate phage/bacteria interactions and the isolation of phages;Using lower temperatures (25–28 °C) than those routinely used in clinical microbiology (30–37 °C) ,36 and lUsing 96- or 384-well microtiter plates for the incubation of a large number of inoculums of bacteria hooks is more convenient. The bacterial suspensions are collected from each well using a multichannel pipette ;v/v) of chloroform to PL at +4 °C (temperature shock) kills the remaining intact bacterial cells, including lyrically phage-infected bacteria, and could thus result in substantially increased phage titers [After incubation, the potential phage lysate (PL) is centrifuged and filtered. There is no necessity for the use of chloroform, as this could reduce the viruses’ infectivity  or inacte titers . Chlorofe titers . In addiThe obtained PL could be used further as the second source for another enrichment BP with different bacteria hooks.Bacteria hooks consisting of working host strains, i.e., strains that have already been adapted/approved for phage propagation/production, speed up downstream phage adaptation/training procedures. Newly isolated phages could, of course, also be propagated and trained in other bacterial strains than the ones used for isolation. A scaled-up version of the PE approach is described by Olsen et al. as part of a high-throughput screening (HiTS) method for phages. They propose using 96-deep-well plates, which allows for the simultaneous handling of a large range of environmental samples (water). One single host is used in each well containing 1.5 mL of water sample, and the method is oriented towards predominantly lytic and easily cultivable phages . An outlIt is considered a disadvantage of the PE method that faster-growing phages will outcompete phages with slower-growing populations , maskingGenerally, the PE lysate is first tested against the bacteria hooks used in the PE method, but it could also be carried out using any other relevant BP, for instance, containing strains from available bacterial culture collections . Differe(i)The “direct spot test” : in which only one dilution of the phage lysate is spotted on bacteria grown directly on solid agar. It is described below;(ii)The “spot test”  (we will(iii)The “lysis profile assay”  or, as wSeveral parallel streaks  of bBacterial suspensions are simply spotted  in a griBacterial suspensions are directly streaked on streaks of phages made on solid agar  we call.In the “direct spot test”, bacteria can be grown either as a series of distinct areas (streaks or spots) or as complete lawns on solid agar (without soft agar overlay). Phage lysates are applied in the areas of expected bacterial growth. Bacteria are commonly applied in three ways:The first two preliminary phage detection approaches allow for the screening of large numbers of BPs and phages. The choice between either of them is a matter of practicality.It is considered that the “spot-on-streak” assay (a variation of the “direct spot test”) does not allow for the evaluation of a possible emergence of bacterial phage resistance . In factAll the previously mentioned methods should be considered as preliminary detection techniques, as they are merely revealing bacterial lysis on agar or in liquid and do not confirm that these are the result of phage activity.4 colony-forming units (cfu)/mL, which will result in sufficient growth to reveal the activity of phages that are present at a low concentration. The “spot-on-streak” assay allows for the application of multiple phage lysates on multiple bacterial strains, at different dilutions, on one plate. Note that bacteria grow slower on a solid agar surface than in broth, which will help phages that are present at lower titers, or with slower reproduction rates, to pace the bacterial growth and reveal themselves. Moreover, when large-size phage virions cannot diffuse [As PE lysates potentially consist of different phage variants at different concentrations, possibly including rare and interesting variants at low titers, it is reasonable to continue evaluating the PE lysates without diluting them. Bacterial suspensions used in the above-mentioned methods should have a minimum concentration of 10 diffuse  in soft  diffuse , thus in diffuse .Pipetting robots could be used for the “spot-testing”-based methods. A rectangular-shaped tray-plate, from SPL life science, for instance, is perfect to perform the spotting and could be fixed on the pipetting robot workstation. The advantage of that plate is that it has nearly the same dimensions (127.94 × 85.50 mm) as a 96-well microtiter plate (127.71 × 85.43 mm), which can be used as a reservoir for the phages that will be spotted. The spotting height should be adjusted correctly to avoid piercing the agar surface or splashing the drop while spotting, and thus generating aerosols and subsequent cross-contamination. In case of the “spot-on-streak” assay, bacterial streaks are pre-prepared, while the “spot-on-spot” method could be performed entirely by the pipetting robot.After visual examination of the lysis zones and interpretation of the preliminary results, several phage/host bacteria combinations are selected to be further submitted to confirmatory methods that are able to reveal true phage plaque formation.Plaque formation is the result of multiple rounds of infection, lysis, and release of progeny , and it While a variety, or the technical modification, of methods are used for plaque formation and enumeration, double agar layer (DAL) methods are the most commonly used.Confirmation of plaque formation;Study of plaque morphology;Enumeration (determination of pfu/mL) of phages.The main reasons for using plaque formation assays are:Phage differentiation/selection;Plaque purification;Phage virulence/lysogeny evaluation procedures.The morphological appearance of the individual plaques is the first parameter that needs to be determined, as it is of great importance for:Plaque diameter;Level of transparency/turbidity of the plaques;Halo formation and size;Motility.The DAL method was independently developed by the Belgian microbiologist André Gratia in 1936 (“Des relations numériques entre bactéries lysogènes et particules de bactériophage”), and by Hershey, Kalmanson, and Bronfenbrenner in 1943 , to be fAnother phage enumeration method described by Kutter et al. , Kropinski, and Mazzocco et al. (“Drop Plaque Assay”) ,52,53 isThe disadvantage of the MD/SP DAL method is that it is not precise enough. For more accurate counting and a perfect comparison of the plaque sizes and morphologies on each strain , the SD/In addition, some studies have shown that particular phages only reveal clear lysis in the first two dilution spots, with no sign of lytic activity in further dilution spots. The reason for this could be an abortive infection, or “lysis from without” , or someFor plaque differentiation and purification, the most commonly used and described method uses phage streaks  on a bacThe distance between the plaques (well isolated discrete plaques);Different dilutions of phage lysate are applied;A certain number of passaging rounds are performed ;Several bacterial host bacterial strains are used;Several growth media are used.To validate the plaque purification method as a confirmatory method, the following criteria need to be considered:For practical convenience, mini petri dishes of 35 mm diameter can be used for plaque formation/passaging assays. It is highly recommended to perform a valid plaque purification procedure before moving on to further characterization and activity evaluation.Bacterial strains for phage host range studies are referred to here as “bacteria kits”. MD/SP DAL is mostly used for the evaluation of EOP . TherefoThe concept of host range or breadth  can be dhttps://www.atcc.org/microbe-products#t=productTab&numberOfResults=24 (accessed on 30 March 2022)) or the Deutsche Sammlung von Mikroorganismen und Zellkulturen  [FDA guidance on antibiotic testing requires the testing of at least 100 bacterial strains, and for some species more than 300 strains, with recent clinical isolates accounting for at least 75% of the strains . Followich 2022)  and inclAt the same time, according to the FDA and some other regulatory bodies for diagnostics , preliminary and confirmatory tests are the main components of systematic qualitative analysis, and this kind of approach needs to be tailored to phage identification, enumeration, and activity evaluation.The phage liquid culturing (PLC) method is considered an alternative approach for phage host range and lytic activity measurement . In addiThe PLC method, or “Appelmans’ method”, was developed in the 1920s by the Belgian surgeon René Appelmans ,62. InitPhage enumeration with phage titer expressed as a dilution factor;Estimation of the multiplicity of infection (MOI) , i.e., tEvaluation of host range and lytic activity ;Expansion of host range after multiple passaging.The Appelmans’ technique can be used for different purposes:Nowadays, the PLC, or Appelmans’ method, is mostly used and described for the study of phage host range and lytic activity in view of translation to the in vivo context. Different interfering/misleading factors may arise when using this method, such as the growth of phage-mutants , the “rePhage-exposed bacterial growth curves have been extensively studied ,70,71,72P. aeruginosa cystic fibrosis bacterial isolates lead to increased pathogen clearance and a lowered resistance evolution as well [Today, the experimental evolution of individual phages or phage mixtures through serial interactions with one or a mixture of host bacteria is the most used approach to extend the phage host range. Several studies performed in the period 1963–1991 describe the benefit of serial passage experiments (SPEs) that allow for molecular and phenotypic evolution in real time . The cha as well .K. pneumoniae [Eastern European researchers, particularly in the Republic of Georgia, used the noted Appelmans’ dilutions method  for passeumoniae .Burrowes et al. designed a 96-well plate formatted for Appelmans’ protocol to analyze the individual phages after every 10 rounds of evolution. They showed that starting with a phage cocktail resulted in a larger host-range expansion than when using individual phages, and based on genomic analysis, they observed a recombinatorial origin for output phages with a broadened host-range .The crucial factors for ensuring a rapid host range extension of phages are (1) the use of a phage mixture from the start, which allows recombination to generate sufficient diversity, and (2) the use of both the original bacterial hosts that had been used for phage propagation and an updated collection of clinical bacterial isolates that are resistant to the given phages, as it is important to produce therapeutically useful phages .While Burrowes et al. state that the Appelmans protocol works predominantly via recombination between phages ,85, MapeSerial passaging for HRE can be performed on agar as well, whenever liquid media are not adequate for demonstration of phage lytic activity. The agar method is more time consuming than the liquid method, but it has the advantage that the obtained phage mixture no longer needs to be processed further for plaque formation .Many studies refer to existing gaps in standardization and validation of assays/methods documenting phage activity and in the translation of their results to in vivo applications ,32,34,87Methods determining phage lytic activity  are baseAs we mentioned earlier, the “direct spot test” and “spot test” methods should only be considered as preliminary  phage detection (sensitivity) tests, as they are not demonstrating plaque formation, while the MD/SP DAL method can be considered as a confirmation test for phage detection as it demonstrates plaque formation. Since the SD/MP DAL method allows for the most precise phage enumeration and plaque morphology characterization, it could be considered as a confirmatory method for phage enumeration and plaque morphology characterization. Note that the MD/SP DAL method is less time-, material-, and labor-consuming as it allows for the analysis of several phages or phage dilutions on one plate. It would be relevant for host range determination and EOP evaluation. We will not provide a detailed discussion of phage culture purification here, as it is beyond the scope of the present review and would deserve a dedicated paper. However, to ensure that a particular phage lysate (newly isolated or evolved) is a single phage particle product and authentic, it first must go through plaque and then culture purification steps. For adequate phage purification, five or more passages should be performed using the “phage T-streaking” method as a preliminary approach, followed by five or more passaging steps using the SD/MP DAL method, as this method allows for full morphological selection and characterization of phage plaques. Once a particular phage is purified (plaque and culture) using an established and validated procedure, the candidate phage can be submitted to further characterization.The PLC method has also been put forward as an alternative approach for phage host range measurement . It is fTM system provides a high-throughput capability (4800 phage assays) [To analyze phage/bacterium population interaction dynamics in a comprehensive manner, it is advised not to use OD measurement, but to measure the conversion of water-soluble tetrazolium salts, which yields a higher sensitivity and dynamic range. For this, the OmniLog assays)  for the The main reason for attempting to standardize phage lytic activity measurements and make them as effective as possible is to be able to correlate phage in vitro traits  with theThe phage liquid culturing (PLC) method is put forward as the best assay to evaluate phage lytic activity ,17,78, iSome relevant phage infection parameters, such as adsorption rate, latent period, and burst size, can be deduced from monitoring phage growth in liquid media . Phage iEvery phage candidate with the potential to be used in the therapy—be it naturally isolated, with or without expanded activity or host range, genetically engineered or not, or used within a ‘one-size fits all’ or broad-spectrum approach —should iTherefore, the question remains as to what should be considered and tested to determine a phage’s potential to reduce the bacterial population at different infection loci.S. aureus rarely expresses its capsular polysaccharides, which are typical for clinical isolates, when they are grown in the laboratory [P. aeruginosa possesses an arsenal of virulence factors enabling it to invade host cells and circumvent host defenses [Certain bacterial determinants are critical for the outcome of phage/bacteria interactions. In vitro and in vivo phage/mixture testing is most often performed using homogeneous bacterial populations grown either on agar, as planktonic cells in liquid culture, or in biofilms. However, the bacterial composition of the infection loci to be treated with phages  usually consists of an assembly of different strains belonging the same or different bacterial species  and exhiboratory . P. aerudefenses , which adefenses , body maThe right phage-bacteria ratio or so-called MOI to achieve complete bacterial lysis over a given period of time in a liquid culture should be determined ,51. The The optimal phage-bacteria ratio is correlated with phage = bacteria growth rates, and the balanced combination of phage-bacteria is the main determinant for the successful reduction/delay of the emergence of phage-resistant bacterial mutants. This optimal phage/bacteria ratio can be used in phage-virulence assays or in vitro and animal models.An appropriate phage mixture or cocktail  is belieFinally, we can conclude that, to date, no validation procedure/format has been developed nor approved by the relevant regulatory authorities for the evaluation, categorization/ranking (preliminary or confirmatory), or documentation of the methods used to assess in vitro and in vivo phage activity in a standardized manner. All of the methods commonly shared and used so far are copied, developed, or modified from manuals and scientific papers, mostly dating from d’Hérelle’s time.The level of phage virulence as a whole (phage therapy capacity)—host detection, host range, phage-bacteria growth rate, phage-bacterial interaction , phage survival/sustainability, adaption to the host, and invading ability—is associated with conditional factors such as patient age and physiology , concentration of bacteria, temperature, and pH at the infection site. Thus, as phage-bacterial interactions are continuously evolving, so is phage virulence. Phage virulence capacity could be enhanced in vitro by implementing a good understanding of phage-bacterial interactions under certain specific conditions (resembling those at the infection loci). In vitro evaluation of phage activity, using standardized and integrated criteria, is bound to provide a valuable support for in vivo applications. Every selected method should be rational, reliable and appropriate in a particular situation, feasible, and cost effective, considering timelines, labor, and material consumption.


Anemia is a major health problem in Saudi Arabia and has multiple etiologies. Many studies have been conducted in Saudi Arabia in specific population groups like school children, adolescents, university students, and females in the reproductive age group, and most have reported high prevalence of anemia. This study was conducted in a specialist hospital in Makkah city and includes all outpatients aged 15 years and above.  To study the burden of anemia among hospital attendees, its stratification based on gender and age, and its severity along with the morphological types of anemia.  This is a study conducted at a specialist hospital in Makkah city and one-month data were collected retrospectively from the laboratory database and include demographic and routine hematological results of complete blood count (CBC).  A total of 21,524 patients were included, out of which 9444 (43.9%) were males and 12020 (56.1%) were females. The overall prevalence of anemia was 38.7% (8339). Prevalence was very high in females, accounting for 68.2% (5689), whereas it was 31.8% (2650) in males. There were 39.6% (3301), 43.9% (3657), and 16.6% (1381) cases of mild, moderate, and severe anemia, respectively. In females, anemia was more prevalent in the age group of 15 to 49, which is considered as the reproductive age group. Microcytic anemia was the most prevalent type observed in this age group, accounting for 40.7% of all anemia cases. Normocytic anemia was more prevalent in the males, accounting for 52%.  Our study showed high prevalence of anemia among the patients attending outpatient departments in a specialist hospital. Females have high prevalence of anemia when compared to male population. Microcytic anemia was the most common anemia type among females and was seen in the 15–49 age group. There is an increase in prevalence of anemia with age for males, whereas, in females, increased prevalence is observed in the reproductive age groups and the anemia prevalence maintained a steady decrease towards the 5th to the 9th decades. Normocytic anemia was more prevalent in the 5th to the 9th decades, indicating that there are more etiologies other than iron deficiency in the causation of anemia. Macrocytic anemia was the least reported anemia type. Anemia of mild and moderate severity was predominant in both genders, although severe anemia showed higher prevalence in females as compared to males. Conclusion. Anemia is highly prevalent in adolescents, adults, and the elderly in Makkah region. The most common cause is thought to be iron deficiency, although other causes are not uncommon. The authorities need to address the problem of prevention and reduction in anemia prevalence by taking effective measures and interventions. Our study showed high prevalence of anemia among the patients attending outpatient departments in a specialist hospital. Females have high prevalence of anemia when compared to male population. Microcytic anemia was the most common anemia type among females and was seen in the 15–49 age group. There is an increase in prevalence of anemia with age for males, whereas, in females, increased prevalence is observed in the reproductive age groups and the anemia prevalence maintained a steady decrease towards the 5th to the 9th decades. Normocytic anemia was more prevalent in the 5th to the 9th decades, indicating that there are more etiologies other than iron deficiency in the causation of anemia. Macrocytic anemia was the least reported anemia type. Anemia of mild and moderate severity was predominant in both genders, although severe anemia showed higher prevalence in females as compared to males.  Anemia is a global health issue affecting a quarter of the global population . It affeWHO has reported global prevalence of 30.2% among nonpregnant women (15–49.99 years) and about 33% in Asia and 44.4% in Africa. Among men (aged 15–59.99 years) and the elderly (≥60 years), the global anemia prevalence is 12.7% and 23.9%, respectively [Anemia is a common problem in low- and middle-income countries, particularly among adolescent females, women of reproductive age, pregnant women, and children. Anemia is expected to be reduced in women of reproductive age (15–49 years) by 50 percent by 2025, according to the second of the world's six global nutrition objectives. Given the fact that anemia affects half a billion women of reproductive age around the world, eliminating anemia is essential for the health as well as their economic production. According to the Global Health Observatory, anemia prevalence among women of reproductive age ranged from 9.1 percent in Australia to 69.6 percent in Yemen in 2016 .According to reports, anemia is a significant health burden in the Gulf countries, with a high frequency of anemia in females between the ages of 17 and 24 years, as well as males, being reported [According to the findings of a study conducted on Saudi women aged 15–49 years, anemia was found in 40% of the participants . Many stThis study was done using the patient's data collected retrospectively for one month from a specialist hospital in Makkah city. The data were collected from the laboratory database and include demographic and routine hematological results of complete blood count (CBC) performed on a fully automated hematology analyzer. Hematological data from all patients aged 15 years and above and attending the routine outpatient departments over a period of one month from January 1, 2019, to January 31, 2019, were included for analysis.The hemoglobin cut-off (g/L) for the diagnosis of anemia and its categorization based on severity into mild, moderate, and severe anemia was done as per WHO recommendation as shown in P value <0.05 was considered statistically significant. Continuous variables were expressed as mean ± standard deviation and categorical data were presented as median and interquartile range (IQR). Odds ratios (ORs) and 95% confidence intervals were obtained by logistic regression to determine the impact of age on anemia.Statistical Analysis was done using IBM SPSS Statistic . The specialist hospital lab receives between 400 and 500 blood samples for routine hematological tests every day from patients attending the outpatient clinics. A total of 21,524 patients were included, out of which 9444 43.87%) were males and 12020 (56.2%) were females . The mea.87% wereP < 0.001), 1.011 , and 1.013 , respectively. The OR for microcytic anemia was 0.984  and for macrocytic anemia it was 1.014 .All the anemia cases were categorized into microcytic, normocytic, and macrocytic anemia based on the reference cut-off for mean corpuscular volume (MCV). Microcytic anemia was the most prevalent type among females, whereas normocytic anemia was more prevalent in the males, accounting for 52% of the anemia cases in males Tables  and 5. TThis study was done using the patient's data collected retrospectively for one month from a specialist hospital in Makkah city. In this study, we attempted to analyze the anemia prevalence among the outpatient attendees in various speciality departments. Anemia is a complex disease with nutritional and nonnutritional variables and mechanisms at play. Saudi Arabia, along with the Gulf Arab countries and others, is located in the eastern Mediterranean. In the eastern Mediterranean region, anemia prevalence ranges from 22.6 percent to 63 percent among pregnant women and is 69.6 percent among women of reproductive age . Anemia Owaidah et al.  describeNumerous risk factors for the development of IDA in Saudi women of reproductive age have been identified . DietaryThe cause of anemia in the senior population must be determined, and fresh studies must be conducted to investigate it. Consumption of tea, which contains a high concentration of polyphenols, has been linked to anemia in the elderly. Polyphenols block nonheme iron absorption. Tea drinking is a widespread ritual in Saudi Arabia and is typically drunk before and after meals , 22. AccThe World Health Assembly (WHA) has designated anemia reduction as a global dietary priority for 2025 . There iOur study has some limitations. We included patients who visited a hospital, which may have inflated the numbers, but we excluded hospitalized patients. However, our findings are consistent with those of other research conducted in Saudi Arabia.Our study showed high prevalence of anemia among the patients attending outpatient departments in a specialist hospital. Females have high prevalence of anemia when compared to male population. Microcytic anemia was the most common anemia type among females and was prevalent in the 15–49 age group considered as the reproductive age group, whereas normocytic anemia was more common among the male gender. Anemia of mild and moderate severity was predominant in both genders, although severe anemia showed higher prevalence in females as compared to males. The authorities need to address the problem of prevention and reduction in anemia prevalence in Saudi Arabia by taking effective measures and interventions.