Genomic characterization of APEC phages and evaluation of the efficacy in reducing the loads of APEC O78 infections in chickens

Introduction: The resistance of avian pathogenic Escherichia coli (APEC) poses a serious challenge to the control of bacterial diseases in the poultry industry. Identification of useful phages as alternatives to antibiotics for APEC O₇₈ is a priority.

Methods: The phage LQ5 was isolated from the contents of the chicken intestines. Whole-genome sequencing was performed using the Illumina NovaSeq 2500 platform, and then bioinformatics analysis was conducted on the genome. The application effect of LQ5 in the O₇₈ infection model of chickens was systematically evaluated.

Results: The phage LQ5 was identified as a member of Myoviridae by electron microscopy. Whole-genome sequencing showed that phage LQ5 is a double strand DNA virus with a genome of 171,908 bp containing active components, such as endolysin, holin lysis mediator. Comparison of the bacterial load of APEC in chicken liver and spleen tissue in samples treated with phage LQ5 and Amoxicillin showed that the phage LQ5 reduced the bacterial load compared with the antibiotic.

Discussion: These results have enriched the information of the phage gene bank for APEC, laying the foundation for the development of targeted phage biocontrol agents against the APEC O₇₈ strain.

1 Introduction

Avian pathogenic Escherichia coli (APEC) causes colibacillosis, a severe systemic disease in poultry. This disease manifests as airsacculitis, pericarditis, perihepatitis, and septicemia, resulting in substantial global economic losses (de Oliveira et al., 2021; Dziva and Stevens, 2008). APEC comprises over 180 serogroups, with serotypes O₁, O₂, and O₇₈ being the most prevalent and clinically significant in chickens (Halfaoui et al., 2017). An analysis of 189 studies on APEC reveals a wide distribution of common O serotypes, with O₇₈ being the most prevalent (16%), followed by O₂ (10%) and O₁₁₇ (8%) (Jhandai et al., 2024).

APEC is one of the extraintestinal E. coli, which may cause the spread of zoonotic diseases through contaminated eggs (Abdelhamid et al., 2024). The E. coli infections in the neonatal poultry are being characterized by septicemia. The pathogenicity of APEC is mediated by a diverse arsenal of virulence factors that facilitate colonization, immune evasion, and systemic invasion. Genomic analyses highlight the critical role of adhesins (such as Type-I and P fimbriae), iron acquisition systems (e.g., aerobactin), and secretion systems (notably the Type VI Secretion System, T6SS, which is highly prevalent) in its success (World Health Organization, 2006; Hong et al., 2016). These factors enable APEC to infect extra-intestinal tissues, including the lungs, liver, heart, and brain, making it a formidable pathogen.

Current control strategies rely heavily on antibiotics and biosecurity measures. However, the overuse and indiscriminate application of antibiotics in the poultry industry have fueled the emergence of multidrug-resistant APEC strains (Naghizadeh et al., 2019). A review of 15 (35.71%) studies about E. coli reported 2,269 (35.59%) MDR strains, with 763 (11.97%) strains being ESBLs (extended-spectrum β-lactam) and 82 (1.29%) being CREs (carbapenem-resistant Enterobacteriaceae) (Vulcanescu et al., 2024). This practice poses a severe threat to public health, as resistance genes can transfer via mobile genetic elements to zoonotic bacteria, compromising the efficacy of antibiotics used in human medicine. And a study shows the bacteriphage have the greater potential in the alternative management of colistin-resistant E. coli infections (Zhu et al., 2024). Thus, more and more bacteriphage need to develop effective alternatives to antibiotics for controlling APEC infections. Nevertheless, infections in poultry at veterinary clinical can lead to food safety in the food chain, the main methods of controlling contamination by foodborne pathogens often involve the application of antimicrobial agents, which are now becoming less efficient. There is a growing need for the development of new approaches to combat these pathogens, especially those that harbor antimicrobial resistant and virulent determinants (Oluwarinde et al., 2023).

Bacteriophages (phages), which specifically infect and lyse bacteria, offer a viable therapeutic alternative. The principal advantage of phage therapy lies in its high specificity, enabling it to target pathogenic bacteria while sparing the beneficial host microbiota, which is a frequent limitation of broad-spectrum antibiotics (Qin et al., 2024; Tawakol et al., 2019). Both domestic and international research has confirmed the potential of phages in combating multidrug-resistant APEC. Zhang et al. (2025) found that phage YP6 can effectively lyse bacterial strains and inhibit biofilm formation. In a parallel development, a foreign study (Norambuena et al., 2025) demonstrated that AC-01, a cocktail preparation composed of four phages, exhibited lytic activity against over half (56.3%) of the tested bacterial strains. These findings collectively indicate that phage therapy is expected to become an effective approach for preventing and controlling multidrug-resistant APEC infections. Many studies have focused on phage therapy against APEC in general, but the high serotype diversity demands a targeted approach. Serotype O₇₈ remains one of the most dominant and virulent strains circulating globally. However, the development of specifically tailored and highly effective phage cocktails against this key serotype is underexplored. Furthermore, a comprehensive evaluation that combines detailed in vitro characterization of phage virulence factors (like host range and kinetics) with robust in vivo efficacy data in animal models is essential for translating phage therapy into practical applications.

This study isolated and characterized a novel phage exhibiting potent lytic activity against APEC O₇₈. We systematically evaluated its efficacy both in vitro and in a chicken challenge model. These findings establish a foundation for developing a targeted, phage-based biocontrol agent against APEC O₇₈ isolates. This approach presents a potential strategy to mitigate economic losses and reduce reliance on antibiotics in poultry production.

2 Materials and methods

2.1 Background of bacterial strains

The host bacteria were 10 strains of E. coli of serotypes O₁, O₂ and O₇₈ isolated from chicken livers were stored in the Hubei Academy of Agricultural Sciences, Wuhan, China. ACN17 (O₇₈ serotype) was used for phage isolation, and all strains were used for lytic spectrum determination. All strains were cultured overnight in Eosin Metylene Blue (EMB) (Qingdao Haibo Com Ltd., China) at 37 °C. A loopful of each strain was cultured in Luria-Bertani (LB) broth (Qingdao Haibo Com Ltd.) at 37 °C. ACN17 was resistant to gentamicin, tobramycin, trimethoprim-sulfamethoxazole, chloramphenicol by BD-Phoneix test, and stored in the Hubei Academy of Agricultural Sciences.

2.2 O₇₈ bacteriophage isolation and purification

Intestinal contents samples of 220-day-old laying hens were collected from poultry farms in Hubei province, to isolate specific phage for the APEC O₇₈ serotype. Samples were resuspended in SM buffer overnight at 4 °C, then centrifuged for 10 min at 10,000 × g. Each supernatant was passed through a 0.22 μm filter (Millipore, Billerica, MA, USA) to remove bacteria. The filtrate was incubated with the bacteria for the night and then centrifuged for 10 min at 10,000 × g to remove the bacteria. A total of 100 μL of each newly cultured indicator strain was mixed with 5 mL of 0.5% LB soft agar at 50 °C and then poured on the surface of a plate of prepared 1% LB agar. Detect the presence of phages using a double-layer plate, if there are transparent spots on the plate, it indicates the presence of lytic phage (Chen et al., 2020). The phage LQ5 was purified using the double-layer method and was purified five times. Purified phage LQ5 against the 10 E. coli indicator strains included three strains of O₁, three strains of O₂, and four strains of O₇₈ (Table 1), as determined by the spot method (Kropinski et al., 2009). Twenty microliters of (10⁸ plaque forming units, PFU) of purified phage LQ5 was placed on a double-layer plate and the plates were incubated for 14 h at 37 °C.

Table 1

Table 1. Lytic activity of phage LQ5 against tested strains of APEC.

2.3 Transmission electron microscopy (TEM) of phage LQ5

The morphology of phage LQ5 was examined by TEM (Zou et al., 2023). Purified phage LQ5 suspensions (approximately 10⁹ PFU/mL) were resuspended in 0.1 mol/L ammonium acetate and fixed on carbon-coated grids. Following staining with 2% w/v phosphotungstic acid (PTA), each grid was observed by TEM using a model HT-7700 transmission electron microscope (Hitachi High-Tech Co., Ltd., Tokyo, Japan).

2.4 Extraction of DNA and whole genome sequencing

The genomic DNA (gDNA) of phage LQ5 was extracted by the phenol-chloroform method (Jin et al., 2023). The phage gDNA was sequenced using the Illumina NovaSeq 2,500 platform by Shanghai Personalbio Technology Co., Ltd. Bcl2 fastq (v2.17.1.14) software was used for preliminary quality analysis to obtain raw sequencing data. We used fastp to filter and quality control the raw sequencing data obtained, cut adapters, remove low-quality reads, high-n ratio reads, and get clean reads (Chen et al., 2018). The clean reads were de novo assembled using the metaSPAdes software (Nurk et al., 2017). Different k-mer lengths were selected for testing and the best assembly result was obtained. Then, the clean reads were aligned to the assembled genomic sequence using the bwa software to calculate the coverage (Li and Durbin, 2009). Prediction of protein-coding genes in the genome and phage LQ5 open reading frames (ORFs) were accomplished using GeneMarkS software¹. The protein sequence encoded by the gene was compared with the protein sequence in the database using Diamond BLASTp to infer the function of the hypothetical protein. At the same time, Bacterial and Viral Bioinformatics Resource Center (BV-BRC)² was used for protein functional annotation. Based on the CARD resistance gene database³ was performed to predict antibiotic resistant genes (ARG) were present. VFDB data repository⁴ was performed to predict whether phage habored virulence gene. The phylogenetic tree of the terminase large subunit was constructed using the neighbor-joining method in MEGA 7 (Moon et al., 2025). The tree was drawn to scale and the units used to infer that the evolutionary distance of the phylogenetic tree were the same as the branch length. The P distance method was used to calculate the evolutionary distance. Easyfig 2.2.5⁵ was used to draw genome-wide collinearity.

2.5 Evaluation of the efficacy of phage LQ5 for APEC O₇₈ infections in chickens

The study was approved by the Ethics Committee of the Hubei Academy of Agricultural Sciences (Approval Date: 10 March 2025; Approval Code: 5/2025). And live chickens were humanely euthanized via intravenous injection at the respective time points designated for each experimental group.

To ensure 25 hatchlings, 40 specific-pathogen-free (SPF) chicken embryos were incubated in an incubator maintained at optimal temperature and humidity conditions. Subsequently, 25 healthy one-day-old chicks were transferred to isolators with adlibitum access to feed and water to evaluate the effect of phage LQ5 (Nicolas et al., 2023). 25 healthy one-day-old chicks were divided into five groups (n = 5 per group): group 1 (prevention group, administered before infection), group 2 (treatment group, administered after infection), group 3 (amoxicillin group), group 4 (model group), and group 5 (control group). In the first day, 5 chicks in group 1 (prevention group) were orally administered phage LQ5. Groups of 2, 3, and 4 treated wih phage LQ5, amoxicillin, and phosphate buffered saline (PBS), respectively. In the third day, groups of 1, 2, 3, and 4 were challenged intratracheally with 10⁸ colony forming units (CFU) of APEC O₇₈. The group 5 were as negative control, with only PBS being administered. In the 4th and 5th days, groups 2 and 3 were orally administered with phage and amoxicillin, respectively. The medication used during the amoxicillin group was commercial amoxicillin, with the veterinary drug code 120191199. It was used in accordance with the instructions. The concentration of the phage LQ5 was 10⁹ PFU/mL. Livers and spleens from the five groups were aseptically removed at 10 days post-infection (dpi) to determine the load of APEC O₇₈ (Galal et al., 2021). Briefly, 1 g of liver and spleen of each group were suspended in PBS and homogenized with an Omnimixer homogenizer. Tissue homogenates were serially diluted 10-fold in PBS, and 100 μL of each dilution was plated onto MacConkey-lactose agar plates for bacterial enumeration. The plates were incubated at 37 °C for 24 h, after which the number of pink colonies was counted and expressed as CFU per gram of tissue. Hematoxylin and eosin (H&E) staining was used to assess the injury in different tissues of five groups. The description of organizational changes was based on the book “Pathological Basis of Veterinary Diseases (5th Edition)”, ISBN: 978-7-109-20098-2 (Figure 1).

Figure 1

Figure 1. Animal experiment grouping.

2.6 Statistical analysis

One-way analysis of variance (ANOVA) was used to evaluate the difference between the experimental and control groups. GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA) was used toplot the data. p < 0.05 was considered statistically significant.

3 Results

3.1 Isolation, morphology and host range of phage LQ5

Phage LQ5 was isolated from the intestinal contents of laying hens using the double-layer agar method, with APEC O₇₈ ACN17 strains serving as the host. The purified phage LQ5 formed several transparent plaques on the plate, with approximate diameters of 2 mm (Figure 2A). TEM revealed that phage LQ5 belongs to the Myoviridae family (Figure 2B). The lytic activity of LQ5 to 10 APEC strains is shown in Table 1. LQ5 exhibited lytic activity exclusively against O₇₈ APEC serotypes and showed no infectivity toward O₁ or O₂ serotypes.

Figure 2

Figure 2. (A) Isolation and purification of O₇₈ phage LQ5. (B) Transmission electron micrograph of phage LQ5. The scale is 50 nm.

3.2 Genome analysis of phage LQ5

The gene map of phage LQ5 was shown in Figure 3. Phage LQ5 is a double-stranded DNA phage with a genome length of 171,908 bp and a GC content of 39.52% (GenBank: OR677401). The genome sequence of LQ5 was compared online by BLASTN. The phage with the highest genome homology (98.60%) was E. coli phage WG01 (GenBank: KU878968.1). Multiple genome alignments and phylogenetic tree analyses of phage LQ5 and E. coli phage WG01, 005, C6, KIT01, MX01, PSD2002, VR5, and FP43 are shown in Figure 4. Easyfig 2.2.5 (see text footnote 5) was used to map the genome-wide collinearity ratio. The collinearity of phages WG01 and LQ5 was extremely high (Figure 5). Phage WG01 was isolated from Nanjing, Jiangsu Province, China. Some genes were located in different positions on the DNA chain, which might be a phenomenon of gene rearrangement that occurs in phages in order to enhance diversity and adaptability.

Figure 3

Figure 3. Genome map of phage LQ5 generated by CGView. The regions in green represent the distribution of the coding sequence (CDS) region and the arrows indicate the direction of transcription. The total GC content (39.52%) is indicated in black, while the inner ring with blue and purple histograms indicates GC skew. For clarity, the hypothetical protein is not described on the map.

Figure 4

Figure 4. Cluster analysis of APEC O₇₈ phage LQ5. The phylogenetic tree was constructed based on the neighbor-joining method of the terminase large subunit.

Figure 5

Figure 5. Genome homology analysis of phage LQ5 with WG01. The green arrow represents the open reading frames. The color gradient represents the level of nucleotide identity between the phage genomes.

The results of protein functional annotation using BV-BRC were consistent with those of GeneMarkS. Protein analyses of phage LQ5 are presented in Table 2. The clarified function of the proteins provide a theoretical basis for subsequent studies of functional proteins of phages. The phage genome consists of four known gene cluster modules. DNA replication and modification modules include DNA helicase (ORF261) and DNA polymerase (ORF227). The DNA packaging module includes terminase large subunit (ORF108) and terminase small subunit (ORF109). The lysis module includes lysozyme (ORF158) and holin (ORF23). Finally, structural proteins include tail fiber protein (ORF62), baseplate protein (ORF117), capsid protein (ORF94) and scaffold protein (ORF101). ORF100 encodes the phage principal prtein. ORF130 encodes the main tail fiber assembly protein of phage, while ORF62 encodes the tail fiber of phage. Phage LQ5 has a binary lysis system composed of holin and lysozyme, which can specifically destroy the cell membrane and cell wall of bacteria, respectively, with a completely different mechanism of action from antibiotics (Washizaki et al., 2016). Glutaredoxin (ORF202) was annotated in LQ5. The phylogenetic tree of LQ5 was constructed based on the nucleotide sequence of the terminase large subunit (ORF108). The tail fiber protein was responsible for the specific initial recognition of host bacteria and can be a potential biological cognitive element for detecting bacteria (Wintachai et al., 2024). No phage, transposase, excision enzyme homology, and repressor was predicted in the LQ5 genome. According to the above sequencing results, phage LQ5 was considered a novel phage.

Table 2

Table 2. Analysis of main proteins of phage LQ5.

3.3 Efficacy of phage LQ5 concerning bacterial load in chickens and survival

3.3.1 The survival rate of chickens

We used the one-day SPF chicken model to study the effect of phage on APEC O₇₈ ACN17 infection in chickens. The five groups were compared concerning the content of E. coli O₇₈ in liver and spleen. The groups of model, treatment, prevention, and amoxicillin displayed survival rates of 0, 60, 80, and 80%, respectively (Figure 6).

Figure 6

Figure 6. Survival rates between different groups.

Survival of the different treatment groups was assessed at 10 days post-infection (10 dpi). The model group, challenged intratracheally with 10⁸ CFU of APEC O_78. The control group was treated with an equivalent dose of PBS; The prevention group received phage prophylaxis 24 h prior to bacterial challenge, whereas the treatment group received phage therapy at 1 day post-infection (1dpi), following an intratracheal challenge with 10⁸ CFU of APEC O₇₈. The amoxicillin group was received amoxicillin therapy at 1 day post-infection (1dpi). The control group maintained a 100% survival rate throughout the observation period (days 0–10). The prevention group and the amoxicillin group had a survival rate of around 80%. The treatment group maintained a survival rate of around 60%. The model group experienced a sharp decline in survival rate after day 4, with a survival rate of approximately 0 around day 5.

3.3.2 The bacterial load in the liver and spleen

We used the bacterial loads in the liver and spleen to evaluate the efficacy of the different treatment groups. Compared with the model group, the bacteria loads in spleens in the groups of treatment, prevention, and amoxicillin have significant difference, especially in liver (p < 0.0001) (Figure 7). The results show that the bacterial load in the spleen of the model group is significantly higher than that of the prevention group (***p < 0.0001), treatment group (**p < 0.001), and amoxicillin group (**p < 0.001). There is no significant difference among the prevention group, treatment group, and amoxicillin group (Figure 7A). The results show that the bacterial load in the liver of the model group is significantly higher than that of the prevention group (***p < 0.0001), treatment group (***p < 0.0001), and amoxicillin group (***p < 0.0001). The bacterial load in the prevention group is higher than that in the treatment group and amoxicillin group, and there is no significant difference between the treatment group and amoxicillin group (Figure 7B).

Figure 7

Figure 7. Bacterial counts of liver and spleen. (A) The number of bacteria in the spleen. (B) The number of bacteria in the liver.

The x-axis represents different groups (prevention, model, treatment, amoxicillin), and the y-axis represents (CFU/mL tissue). All samples were collected at 7 dpi (7 days post-infection), and the data are represented by the mean (CFU/mL) of samples from each group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

3.3.3 Morphological observation of the different tissue

Histology analysis revealed some traits in the five tissue types. In the model group, many sites of bleeding were evident in liver sinusoids and cerebral glial membrane; with many red blood cells in the medulla of the spleen, the alveoli of the lungs (along with a widening of the alveolar space), the lungs, the liver. No obvious pathological changes were observed in the other groups. Compared with the model group, the preventive and treatment groups displayed only a small amount of bleeding, while the antibiotic group had more severe symptoms than the preventive and treatment groups. Antibiotics and phages have similar effects in reducing the bacterial load in the liver and spleen. Pathological section results showed that antibiotics and phages have similar effects in reducing the bacterial load in the liver and spleen, and phages have a better protective effect on the liver and spleen tissues (Figure 8).

Figure 8

Figure 8. Histology of chicken tissue sections after H&E staining at 200 × magnification. Histology of chicken tissue sections (heart, liver, spleen, lung, and brain) was observed after H&E staining at 200 × magnification.

The groups include control, amoxicillin, model, prevention, and treatment, with arrows indicating lesions. The control group showed no obvious lesions, with normal tissue structures. Lesions (such as inflammation and tissue damage, indicated by arrows) were observed in the heart, liver, spleen, and lung tissues of the amoxicillin group, model group, and prevention group. The model group had relatively more obvious lesions, while the amoxicillin group and prevention group had lesions to varying degrees. The Treatment group had few lesions in each tissue, close to normal. Interestingly, among all the groups, only the Model group showed vascular cuffing lesion in the brain tissue.

4 Discussion

Phages specifically target pathogenic bacteria without harming beneficial microorganisms (Zhao et al., 2025; Xu et al., 2025). The primary pathogens responsible for intestinal diseases in animals are E. coli, Salmonella, and Clostridium perfringens, with colitis being the most frequent manifestation (Hegarty, 2025). A significant challenge in prevention and treatment is the widespread resistance to first-line antibiotics, necessitating the identification of suitable alternatives such as phage therapy (Antimicrobial Resistance Collaborators, 2022).

Phage LQ5 and vB_EcoM_APEC both belong to the Myoviridae family and are sensitive to APEC O₇₈ (Deng et al., 2021). The characteristics of phage such as MOI, pH tolerance and temperature stability will be reflected in another article about the mechanism of phage resistance. Functional annotation of phage genomes and analysis of their roles in the phage life cycle are essential to determine aspects such as phage interaction, replication, infection of bacteria, coevolution, and host range (Tokodi et al., 2025; Doekes et al., 2021; Holtappels et al., 2023). Phage tail fiber protein can be modified by natural evolution or genetic engineering, including homologous recombination, synthetic biology and directed evolution, to expand their host range or improve their specific recognition ability (Filik-Matyjaszczyk et al., 2025).

The improper use of antibacterial drugs in poultry farming is one of the reasons for the emergence of antimicrobial resistance (AMR) in poultry production. Antibiotic resistant bacteria caused a 1.27 million human deaths in 2019 globally (Iskender and Soyer, 2023). These strategies include phages (Mao et al., 2023) herbal medicines (Zhang et al., 2020), probiotics (Zhang et al., 2023), synthetic CpG oligodeoxynucleotide (Gunawardana et al., 2022). Phages have a positive preventive and therapeutic effect in combating and preventing the drug resistance of APEC strains. Phages reduced the mortality rate of one-day-old chickens by 20 and 30%, respectively, (in the treatment group and the prevention group) (Jhandai et al., 2024). Other studies reported significantly reduced growth of two kinds of APEC treated with phage compared to a control group not treated with phage. PEC9 was a siphovirus-like phage, which lysed 9/20 O₁ and 6/20 O₂ serotypes of E. coli (Lau et al., 2010). These findings implicate PEC9 as having an important role in increasing survival rate, and reducing bacterial loads and liver lesions in APEC infections. Researchers identified vB_EcoM as a member of Myoviridae, with lytic activity for the APEC O₇₈ sereotype (Balcao et al., 2022). In contrast, purified endolysin displayed broad-spectrum lytic activity for the O₇₈, O₁₅₇ sereotypes of APEC, as well as Klebsiella, Salmonella, Shigella, and Yersinia strains (Yao et al., 2023). These findings highlight the importance of evaluating phage with amoxicillin concerning bacteria loads. The preventive and therapeutic results of phage LQ5 are similar to those of antibiotics, providing a reference for the clinical application of phages and offering an alternative to antibiotics for the prevention and control of E. coli O78 infection.

Infection rates ranged from 20 to 85.7% when each phage was used alone and from 78.6 to 88.9% when antibiotics were used (El-Meihy et al., 2024). The mice that received phage therapy did not develop pneumonia caused by multi-drug resistant Staphylococcus aureus. This can be used as a basis for exploring the application of phage therapy in treating Staphylococcus aureus infections in blood (Oduor et al., 2016). In the model of Pseudomonas aeruginosa sinusitis, the safety and effectiveness of phage cocktail in vivo provide a basis for the application of phage (Fong et al., 2019). Studies have shown that in the first stage under bioimaging, the liver and spleen can observe the most obvious bacterial distribution, and tissue lesions in the lungs are more typical, providing reference for our subsequent assessment of bacterial load, thus making the lungs more convincing as an important organ and as a progressive regulator of colisepticaemia (Abdelhamid et al., 2024). The application of these phages has indeed greatly enriched the knowledge and practice of phage safety.

In this study, we successfully isolated a novel APEC phage LQ5, which can specifically lyse the O₇₈ serotype of E. coli. Phage LQ5 reduced the APEC counts in the liver and spleen of chickens, indicating protective effects of chickens infected with APEC O₇₈. Importantly, these results are consistent with previous reports (Deng et al., 2021; Moreno et al., 2025). Phage LQ5 exhibited antibacterial activity comparable to amoxicillin against the O₇₈ serotype. These results further support the potential of phages to reduce antibiotic use in poultry production.

5 Conclusion

This study evaluated the efficacy of phage LQ5 against E. coli O₇₈ infection in chicks. Phage LQ5 increased the survival rate of chicks infected with E. coli, reduced the bacterial load in the spleen and liver, and alleviated the pathological changes in tissues. This study provides a theoretical basis for the use of phages in the prevention of E. coli in chicks, and offers a strategy for the clinical prevention and treatment of colibacillosis. The limitations of this study are the insufficient research on the structure and function of the phage receptor binding protein and the unclear mechanism of host recognition, which will be carried out in future research.

Data availability statement

The data presented in this study are publicly available. The data can be found at: https://www.ncbi.nlm.nih.gov/nuccore/OR677401.

Ethics statement

The animal study was approved by Ethics Committee of Institute of Animal Science and Veterinary Medicine, Hubei Academy of Agricultural Sciences. The study was conducted in accordance with the local legislation and institutional requirements.

Author contributions

QLu: Data curation, Writing – original draft. XJ: Software, Writing – original draft. ZW: Investigation, Writing – review & editing. RZ: Investigation, Writing – review & editing. YG: Investigation, Supervision, Writing – review & editing. QH: Resources, Writing – review & editing. WZ: Formal analysis, Writing – review & editing. TZ: Project administration, Writing – review & editing. QLuo: Conceptualization, Funding acquisition, Writing – review & editing.

Funding

The author(s) declared that financial support was received for this work and/or its publication. As the projects of the Key Projects of Hubei Natural Science Foundation (2021CFA019), and the Key Research and Development Project of Hubei Province 2022BBA0055 have completed, we need change the above two projects into Hubei Province Modern Agricultural Industry Technology System (2025HBSTX4-04) and Wuhan technology commissioner (2024111203060843).

Acknowledgments

The authors would like to thank members of their laboratory for their helpful and constructive advice.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that Generative AI was not used in the creation of this manuscript.

Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Footnotes

^1.^http://topaz.gatech.edu/Genemark/

^2.^https://www.bv-brc.org/

^3.^https://card.mcmaster.ca/

^4.^http://www.mgc.ac.cn/VFs/

^5.^http://mjsull.github.io/Easyfig/

References

Abdelhamid, M. K., Hess, C., Bilic, I., Glosmann, M., Rehman, H. U., Liebhart, D., et al. (2024). A comprehensive study of colisepticaemia progression in layer chickens applying novel tools elucidates pathogenesis and transmission of Escherichia coli into eggs. Sci. Rep. 14:8111. doi: 10.1038/s41598-024-58706-3,

PubMed Abstract | Crossref Full Text | Google Scholar

Antimicrobial Resistance Collaborators (2022). Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet 399, 629–655. doi: 10.1016/S0140-6736(21)02724-0,

PubMed Abstract | Crossref Full Text | Google Scholar

Balcao, V. M., Belline, B. G., Silva, E. C., Pffb, A., Baldo, D. A., Amorim, L. R. P., et al. (2022). Isolation and molecular characterization of two novel lytic bacteriophages for the biocontrol of Escherichia coli in uterine infections: in vitro and ex vivo preliminary studies in veterinary medicine. Pharmaceutics 14:344. doi: 10.3390/pharmaceutics14112344,

PubMed Abstract | Crossref Full Text | Google Scholar

Chen, Y., Li, X., Wang, S., Guan, L., Li, X., Hu, D., et al. (2020). A novel tail-associated O91-specific polysaccharide Depolymerase from a Podophage reveals lytic efficacy of Shiga toxin-producing Escherichia coli. Appl. Environ. Microbiol. 86:20. doi: 10.1128/AEM.00145-20,

PubMed Abstract | Crossref Full Text | Google Scholar

Chen, S., Zhou, Y., Chen, Y., and Gu, J. (2018). Fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34, i884–i890. doi: 10.1093/bioinformatics/bty560,

PubMed Abstract | Crossref Full Text | Google Scholar

de Oliveira, A. L., Barbieri, N. L., Newman, D. M., Young, M. M., Nolan, L. K., and Logue, C. M. (2021). Characterizing the type 6 secretion system (T6SS) and its role in the virulence of avian pathogenic Escherichia coli strain APEC O₁₈. PeerJ 9:e12631. doi: 10.7717/peerj.12631,

PubMed Abstract | Crossref Full Text | Google Scholar

Deng, S., Xu, Q., Fu, Y., Liang, L., Wu, Y., Peng, F., et al. (2021). Genomic analysis of a novel phage infecting the Turkey pathogen Escherichia coli APEC O₇₈ and its Endolysin activity. Viruses 13:1034. doi: 10.3390/v13061034,

PubMed Abstract | Crossref Full Text | Google Scholar

Doekes, H. M., Mulder, G. A., and Hermsen, R. (2021). Repeated outbreaks drive the evolution of bacteriophage communication. eLife 10:8410. doi: 10.7554/eLife.58410,

PubMed Abstract | Crossref Full Text | Google Scholar

Dziva, F., and Stevens, M. P. (2008). Colibacillosis in poultry: unravelling the molecular basis of virulence of avian pathogenic Escherichia coli in their natural hosts. Avian Pathol. 37, 355–366. doi: 10.1080/03079450802216652,

PubMed Abstract | Crossref Full Text | Google Scholar

El-Meihy, R. M., Hassan, E. O., Alamoudi, S. A., Negm, S., Al-Hoshani, N., Al-Ghamdi, M. S., et al. (2024). Probing the interaction of Paenibacillus larvae bacteriophage as a biological agent to control the american foulbrood disease in honeybee. Saudi J. Biol. Sci. 31:104002. doi: 10.1016/j.sjbs.2024.104002,

PubMed Abstract | Crossref Full Text | Google Scholar

Filik-Matyjaszczyk, K., Matyjaszczyk, I., Ciesielska, M., Szermer-Olearnik, B., Mikolajczyk, K., and Gamian, A. (2025). Phage receptor binding protein and fc fragment fusion enhances phagocytosis of Y. enterocolitica. AMB Express 15:135. doi: 10.1186/s13568-025-01948-9,

PubMed Abstract | Crossref Full Text | Google Scholar

Fong, S. A., Drilling, A. J., Ooi, M. L., Paramasivan, S., Finnie, J. W., Morales, S., et al. (2019). Safety and efficacy of a bacteriophage cocktail in an in vivo model of Pseudomonas aeruginosa sinusitis. Transl. Res. 206, 41–56. doi: 10.1016/j.trsl.2018.12.002,

PubMed Abstract | Crossref Full Text | Google Scholar

Galal, H. M., Abdrabou, M. I., Faraag, A. H. I., Mah, C. K., and Tawfek, A. M. (2021). Evaluation of commercially available aroA delated gene E. coli O₇₈ vaccine in commercial broiler chickens under Middle East simulating field conditions. Sci. Rep. 11:1938. doi: 10.1038/s41598-021-81523-x,

PubMed Abstract | Crossref Full Text | Google Scholar

Gunawardana, T., Ahmed, K. A., Popowich, S., Kurukulasuriya, S., Lockerbie, B., Karunarathana, R., et al. (2022). Comparison of therapeutic antibiotics, probiotics, and synthetic CpG-ODNs for protective efficacy against Escherichia coli lethal infection and impact on the immune system in neonatal broiler chickens. Avian Dis. 66, 165–175. doi: 10.1637/aviandiseases-D-22-00011,

PubMed Abstract | Crossref Full Text | Google Scholar

Halfaoui, Z., Menoueri, N. M., and Bendali, L. M. (2017). Serogrouping and antibiotic resistance of Escherichia coli isolated from broiler chicken with colibacillosis in center of Algeria. Vet. World 10, 830–835. doi: 10.14202/vetworld.2017.830-835,

PubMed Abstract | Crossref Full Text | Google Scholar

Hegarty, B. (2025). Making waves: intelligent phage cocktail design, a pathway to precise microbial control in water systems. Water Res. 268:122594. doi: 10.1016/j.watres.2024.122594,

PubMed Abstract | Crossref Full Text | Google Scholar

Holtappels, D., Alfenas-Zerbini, P., and Koskella, B. (2023). Drivers and consequences of bacteriophage host range. FEMS Microbiol. Rev. 47:38. doi: 10.1093/femsre/fuad038,

PubMed Abstract | Crossref Full Text | Google Scholar

Hong, Y., Thimmapuram, J., Zhang, J., Collings, C. K., Bhide, K., Schmidt, K., et al. (2016). The impact of orally administered phages on host immune response and surrounding microbial communities. Bacteriophage 6:e1211066. doi: 10.1080/21597081.2016.1211066,

PubMed Abstract | Crossref Full Text | Google Scholar

Iskender, I., and Soyer, Y. (2023). Phage therapy against pathogenic Escherichia coli (O₁₀₄:H₄, O₁₅₇:H₇, and O₂₆) strains in irrigation water during garden cress (Lepidium sativum Linn.) vegetation. Foodborne Pathog. Dis. 20, 553–562. doi: 10.1089/fpd.2023.0020,

PubMed Abstract | Crossref Full Text | Google Scholar

Jhandai, P., Mittal, D., Gupta, R., Kumar, M., and Khurana, R. (2024). Therapeutics and prophylactic efficacy of novel lytic Escherichia phage vB_EcoS_PJ16 against multidrug-resistant avian pathogenic E. coli using in vivo study. Int. Microbiol. 27, 673–687. doi: 10.1007/s10123-023-00420-7,

PubMed Abstract | Crossref Full Text | Google Scholar

Jin, X., Sun, X., Wang, Z., Dou, J., Lin, Z., Lu, Q., et al. (2023). Virulent phage vB_EfaS_WH1 removes Enterococcus faecalis biofilm and inhibits its growth on the surface of chicken meat. Viruses 15:1208. doi: 10.3390/v15051208,

PubMed Abstract | Crossref Full Text | Google Scholar

Kropinski, A. M., Mazzocco, A., Waddell, T. E., Lingohr, E., and Johnson, R. P. (2009). Enumeration of bacteriophages by double agar overlay plaque assay. Methods Mol. Biol. 501, 69–76. doi: 10.1007/978-1-60327-164-6_7,

PubMed Abstract | Crossref Full Text | Google Scholar

Lau, G. L., Sieo, C. C., Tan, W. S., Hair-Bejo, M., Jalila, A., and Ho, Y. W. (2010). Efficacy of a bacteriophage isolated from chickens as a therapeutic agent for colibacillosis in broiler chickens. Poult. Sci. 89, 2589–2596. doi: 10.3382/ps.2010-00904,

PubMed Abstract | Crossref Full Text | Google Scholar

Li, H., and Durbin, R. (2009). Fast and accurate short read alignment with burrows-wheeler transform. Bioinformatics 25, 1754–1760. doi: 10.1093/bioinformatics/btp324,

PubMed Abstract | Crossref Full Text | Google Scholar

Mao, X., Wu, Y., Ma, R., Li, L., Wang, L., Tan, Y., et al. (2023). Oral phage therapy with microencapsulated phage A221 against Escherichia coli infections in weaned piglets. BMC Vet. Res. 19:165. doi: 10.1186/s12917-023-03724-y,

PubMed Abstract | Crossref Full Text | Google Scholar

Moon, K., Ryu, S., Song, S. H., Chun, S. W., Lee, N., and Lee, A. H. (2025). Characterization of novel bacteriophages for effective phage therapy against Vibrio infections in aquaculture. J. Microbiol. 63:e2502009. doi: 10.71150/jm.2502009,

PubMed Abstract | Crossref Full Text | Google Scholar

Moreno, D. S., Kuczkowski, M., Korzeniowski, P., Grzymajlo, K., Wozniak-Biel, A., Sliwka, P., et al. (2025). Application of UPWr_E124 phage cocktail for effective reduction of avian pathogenic Escherichia coli in mice and broiler chickens. Vet. Microbiol. 302:110398. doi: 10.1016/j.vetmic.2025.110398,

PubMed Abstract | Crossref Full Text | Google Scholar

Naghizadeh, M., Karimi Torshizi, M. A., Rahimi, S., and Dalgaard, T. S. (2019). Synergistic effect of phage therapy using a cocktail rather than a single phage in the control of severe colibacillosis in quails. Poult. Sci. 98, 653–663. doi: 10.3382/ps/pey414,

PubMed Abstract | Crossref Full Text | Google Scholar

Nicolas, M., Faurie, A., Girault, M., Lavillatte, S., Menanteau, P., Chaumeil, T., et al. (2023). In ovo administration of a phage cocktail partially prevents colibacillosis in chicks. Poult. Sci. 102:102967. doi: 10.1016/j.psj.2023.102967,

PubMed Abstract | Crossref Full Text | Google Scholar

Norambuena, R., Rojas-Martinez, V., Tobar-Calfucoy, E., Aguilera, M., Sabag, A., Zamudio, M. S., et al. (2025). Development of a bacteriophage cocktail with high specificity against high-risk avian pathogenic Escherichia coli. Poult. Sci. 104:105038. doi: 10.1016/j.psj.2025.105038,

PubMed Abstract | Crossref Full Text | Google Scholar

Nurk, S., Meleshko, D., Korobeynikov, A., and Pevzner, P. A. (2017). metaSPAdes: a new versatile metagenomic assembler. Genome Res. 27, 824–834. doi: 10.1101/gr.213959.116,

PubMed Abstract | Crossref Full Text | Google Scholar

Oduor, J. M. O., Onkoba, N., Maloba, F., and Nyachieo, A. (2016). Experimental phage therapy against haematogenous multi-drug resistant Staphylococcus aureus pneumonia in mice. Afr. J. Lab. Med. 5:435. doi: 10.4102/ajlm.v5i1.435,

PubMed Abstract | Crossref Full Text | Google Scholar

Oluwarinde, B. O., Ajose, D. J., Abolarinwa, T. O., Montso, P. K., Du Preez, I., Njom, H. A., et al. (2023). Safety properties of Escherichia coli O₁₅₇:H₇ specific bacteriophages: recent advances for food safety. Foods 12:989. doi: 10.3390/foods12213989,

PubMed Abstract | Crossref Full Text | Google Scholar

Qin, K., Shi, X., Yang, K., Xu, Q., Wang, F., Chen, S., et al. (2024). Phage-antibiotic synergy suppresses resistance emergence of Klebsiella pneumoniae by altering the evolutionary fitness. MBio 15:e0139324. doi: 10.1128/mbio.01393-24,

PubMed Abstract | Crossref Full Text | Google Scholar

Tawakol, M. M., Nabil, N. M., and Samy, A. (2019). Evaluation of bacteriophage efficacy in reducing the impact of single and mixed infections with Escherichia coli and infectious bronchitis in chickens. Infect. Ecol. Epidemiol. 9:1686822. doi: 10.1080/20008686.2019.1686822,

PubMed Abstract | Crossref Full Text | Google Scholar

Tokodi, N., Lobodzinska, A., Klimczak, B., Antosiak, A., Mlynarska, S., Sulcius, S., et al. (2025). Proliferative and viability effects of two cyanophages on freshwater bloom-forming species Microcystis aeruginosa and Raphidiopsis raciborskii vary between strains. Sci. Rep. 15:3152. doi: 10.1038/s41598-025-87626-z,

PubMed Abstract | Crossref Full Text | Google Scholar

Vulcanescu, D. D., Bagiu, I. C., Avram, C. R., Oprisoni, L. A., Tanasescu, S., Sorescu, T., et al. (2024). Bacterial infections, trends, and Resistance patterns in the time of the COVID-19 pandemic in Romania-a systematic review. Antibiotics 13:1219. doi: 10.3390/antibiotics13121219,

PubMed Abstract | Crossref Full Text | Google Scholar

Washizaki, A., Yonesaki, T., and Otsuka, Y. (2016). Characterization of the interactions between Escherichia coli receptors, LPS and OmpC, and bacteriophage T4 long tail fibers. Microbiology 5, 1003–1015. doi: 10.1002/mbo3.384,

PubMed Abstract | Crossref Full Text | Google Scholar

Wintachai, P., Thaion, F., Clokie, M. R. J., and Thomrongsuwannakij, T. (2024). Isolation and characterization of a novel Escherichia bacteriophage with potential to control multidrug-resistant avian pathogenic Escherichia coli and biofilms. Antibiotics 13:1083. doi: 10.3390/antibiotics13111083,

PubMed Abstract | Crossref Full Text | Google Scholar

World Health Organization (2006). Safety evaluation of certain contaminants in food. Prepared by the sixty-fourth meeting of the joint FAO/WHO expert committee on food additives (JECFA). FAO Food Nutr. Pap. 82, 1–778.

Google Scholar

Xu, X., Yang, J., Hao, G., Wang, B., Ma, T., Zhu, S., et al. (2025). Three in one: a multifunctional oxidase-mimicking ag/Mn(3)O(4) nanozyme for colorimetric determination, precise identification, and broad-spectrum inactivation of foodborne pathogenic bacteria. Food Chem. 464:141620. doi: 10.1016/j.foodchem.2024.141620,

PubMed Abstract | Crossref Full Text | Google Scholar

Yao, L., Bao, Y., Hu, J., Zhang, B., Wang, Z., Wang, X., et al. (2023). A lytic phage to control multidrug-resistant avian pathogenic Escherichia coli (APEC) infection. Front. Cell. Infect. Microbiol. 13:1253815. doi: 10.3389/fcimb.2023.1253815,

PubMed Abstract | Crossref Full Text | Google Scholar

Zhang, H., Liu, Z., Fang, H., Chang, S., Ren, G., Cheng, X., et al. (2023). Construction of probiotic double-layered multinucleated microcapsules based on sulfhydryl-modified Carboxymethyl cellulose sodium for increased intestinal adhesion of probiotics and therapy for intestinal inflammation induced by Escherichia coli O₁₅₇:H₇. ACS Appl. Mater. Interfaces 15, 18569–18589. doi: 10.1021/acsami.2c20437

Crossref Full Text | Google Scholar

Zhang, S., Ye, Q., Wang, M., Zhu, D., Jia, R., Chen, S., et al. (2025). Isolation and characterization of a broad-spectrum bacteriophage against multi-drug resistant Escherichia coli from waterfowl field. Poult. Sci. 104:104787. doi: 10.1016/j.psj.2025.104787,

PubMed Abstract | Crossref Full Text | Google Scholar

Zhang, X., Zhao, Q., Wu, C., Xie, Z., Ci, X., Li, H., et al. (2020). Nitrate is crucial for the proliferation of gut Escherichia coli caused by H₉N₂ AIV infection and effective regulation by Chinese herbal medicine Ageratum-liquid. Front. Microbiol. 11:555739. doi: 10.3389/fmicb.2020.555739,

PubMed Abstract | Crossref Full Text | Google Scholar

Zhao, J., Wang, D., Wang, C., Lin, Y., Ye, H., Maung, A. T., et al. (2025). Biocontrol of Salmonella Schwarzengrund and Escherichia coli O₁₅₇:H₇ planktonic and biofilm cells via combined treatment of polyvalent phage and sodium hexametaphosphate on foods and food contact surfaces. Food Microbiol. 126:104680. doi: 10.1016/j.fm.2024.104680,

PubMed Abstract | Crossref Full Text | Google Scholar

Zhu, X., Xiao, T., Jia, X., Ni, X., Zhang, X., Fang, Y., et al. (2024). Isolation and evaluation of bacteriophage cocktail for the control of colistin-resistant Escherichia coli. Microb. Pathog. 197:107056. doi: 10.1016/j.micpath.2024.107056,

PubMed Abstract | Crossref Full Text | Google Scholar

Zou, H., Ding, Y., Shang, J., Ma, C., Li, J., Yang, Y., et al. (2023). Isolation, characterization, and genomic analysis of a novel bacteriophage MA9V-1 infecting Chryseobacterium indologenes: a pathogen of Panax notoginseng root rot. Front. Microbiol. 14:1251211. doi: 10.3389/fmicb.2023.1251211,

PubMed Abstract | Crossref Full Text | Google Scholar