Escherichia coli is a kind of conditional pathogen, typically not causing pathogenic effects on the host under normal circumstances. However, it can cause a variety of infections when there is low host immunity or a compromised gastrointestinal barrier [1,2]. The continuous emergence of multidrug-resistant and fully drug-resistant E. coli poses a serious threat to human life and health [3, 4]. Bacteriophages are viral entities present in the environment that have the ability to infect bacteria, leading to a rapid increase in their population within the bacterial host, ultimately causing bacterial to lyse and die[5]. Originally, they are extensively utilized in the treatment of pathogenic bacterial infections and in the inhibition of pathogen growth in the external environment. Their potential as an antibacterial agent has attracted considerable interest [6]. Numerous studies have demonstrated the efficacy of phages as an adjunct or even alternative treatment for infections caused by multidrug-resistant bacteria [7]. Isolation of more novel phages and elucidation of their genome sequences to accumulate a sufficient number of phage reserves for the preparation of phage cocktails against specific drug-resistant bacteria lays the foundation for phage therapy of infections caused by multidrug-resistant and fully drug-resistant bacteria. In order to develop Escherichia phage reserves, we isolated and identified a novel Escherichia phage GaoY1-9D.
In this study, avian pathogenic E. coli served as the host bacterium. It was cultured at 37°C in Luria-Bertani medium with shaking. Subsequently, Escherichia phage GaoY1-9D was isolated from farm sewage samples using a previously described method [8]. For transmission electron micrograph, we pipetted 20 μL of the purified phage suspension and 20 μL of 2% phosphotungstic acid on the copper mesh, and then observed under the transmission electron microscope (Specifications:JEM-1400). For the one-step growth curve, the host bacterial solution (108 CFU/mL) was mixed 1:1 with the phage (1010 CFU/mL), incubated at 37 °C for 10 min, centrifuged at 8000 rpm for 5 min, and the supernatant was discarded. Sediment was resuspend by 5 mL of LB liquid medium and incubated at 37 °C for 120 min. samples were taken and the phage titer was determined every 5 min. To assess the thermal stability, the phage was underwent treatment in a -70 °C ultra-low temperature freezer, as well as at temperatures of 4 °C, 30 °C, 40 °C, 50 °C, 60 °C, 70 °C, and 80 °C in a metal bath for a duration of 1 hour. Subsequently, the phage titer was determined following gradient dilution. The stability of phages at various pH levels was assessed by preparing buffers with pH values ranging from 1 to 12. 900 μL of buffer was mixed with 100 μL of phage suspension, incubated at 37 °C for 2 h, and then diluted sequentially to measure phage titers. Three sets of parallel experiments were set up for each experiment.
Phage genomic DNA was extracted with phenol, chloroform and resuspended in double-distilled water. Perform whole genome sequencing of phages GaoY1-9D using the DNBSEQ-T10 PE150 sequencing platform (Chengdu Life Baseline Technology Co., Ltd, Chengdu, China). The whole genome sequencing data obtained was followed by filtering and quality control of the raw sequencing data obtained using fastp [9]. Then cut the adapter, remove the low-quality reads, and finally get clean reads. Utilized the Shovill (https://github.com/tseemann/shovill) software to assemble clean reads for denovo assembly, and analyzed the end of phage genome using CheckV [10]. After the genome was annotated with the coding genes and tRNAs using Prokka [11], the annotation results were manually organized by CGView Server [12] (https://cgview.ca/) to create a genome annotation map. Virulence factors were predicted using the VFDB [13] online website (http://www.mgc.ac.cn/cgi-bin/VFs/v5/main.cgi). ResFinder [14] (http://genepi.food.dtu.dk/resfinder) was used to predict the presence of drug-resistant genes on the genome. The phage genomes were submitted to NCBI for BLASTN comparison to identify homologous species. Subsequent to aligning the homologous phage genomes using MAFFT [15], the aligned data files were imported into MEGAX [16] to construct the phylogenetic tree. Phylogenetic analysis was conducted utilizing FastANI [17], and comparative genomic visualization analysis was carried out using Clinker v0.0.23 [18].
Phage GaoY1-9D electron microscopy examination revealed an icosahedral head measuring 60 nm in diameter, along with a tail that was 140 nm long and 15 nm wide (Fig. 1A). The incubation period of GaoY1-9D was about 40 min, followed by a release period of 20 min, and the burst size was 20 PFU/cell (Fig. 1B). Analysis of thermal stability depicted that phage GaoY1-9D was susceptible to high temperatures of 70 °C and 80 °C, resulting in a sharp decline in titer to 0 PFU/mL at both temperatures. Despite this, the phage exhibited robust infectivity within the temperature range of 4-60°C, and its effectiveness persisted even after prolonged storage at -70°C (Fig. 1C). The pH stability analysis found that the phage GaoY1-9D was not resistant to either strong acids or strong bases (Fig. 1D). Its activity was lost in strong acid buffers at pH levels of 1, 2, and 3, as well as in strong alkali buffers at pH 12. GaoY1-9D can tolerate a pH range of 4-10, with a wide range of pH stability, which can meet the needs of therapeutic applications. We determined the host spectrum of the phage GaoY1-9D using laboratory-preserved and isolated E. coli. In addition to infecting its own host E. coli 1-9, it can also infect E. coli 1-25 and E. coli 7-2 (Table S1).
Escherichia phage GaoY1-9D formed transparent empty spots on bilayer plates of avian pathogenic E. coli, suggesting that phage GaoY1-9D was a virulent phage. GaoY1-9D consisted of a circular double-stranded DNA molecule that contained a terminal repeat sequence spanning 127 bp (Fig. 2A). The total length of the genome was 50,368 bp, with a GC content of 46.46%. In the genome, a total of 83 putative protein-coding genes were identified (Table S2), and the phage lacked genes responsible for coding tRNAs. There were only 7 genes which encoded known functional protein (Fig. 2B). The GaoY1-9D was able to encode the tape measure protein (gp28), which correlated with the size of the phage tail and facilitated the transfer of phage DNA into the cytoplasm when infecting the host bacterium. Interestingly, it only encoded the structural protein tail tip protein L (gp30). Furthermore, the GaoY1-9D genome contained numerous hypothetical proteins whose functions remain unidentified, indicating a wide range of diversity within the GaoY1-9D genome. The holin protein was absent in the GaoY1-9D genomes, while endolysin (gp55) was identified. Additionally, genes encoding integrases were notably absent in these genomes. According to the virulence gene prediction software, the genome of GaoY1-9D lacked virulence genes and no genes associated with drug resistance were found.
Phylogenetic tree results showed that Escherichia phage GaoY1-9D and Escherichia phage vB_EcoS_ESCO41 (NC_047820.1) formed a distinct branch (Fig. 3A). Using the BLASTN program at NCBI, the results also showed that Escherichia phage vB_EcoS_ESCO41 was most closely associated with GaoY1-9D (coverage, 69%; identity, 92.98%). The average nucleotide identity (ANI) result between the two phages was 89.93%, with a comparison coverage rate of 69%, indicating GaoY1-9D represent a novel phage. Furthermore, global genome comparison between GaoY1-9D and vB_EcoS_ESCO41 showed that 17 functional regions in phage GaoY1-9D genome cannot be matched to phage vB_EcoS_ESCO41(Fig. 3B). These regions exclusively consisted of hypothetical proteins with unknown functions. In addition to differing in genes related to DNA replication and nucleic acid metabolism, Escherichia phage vB_EcoS_ESCO41 had a tail protein difference over GaoY1-9D. Moreover, Escherichia phage vB_EcoS_ESCO41 contains a unique double zinc ribbon protein distinct from GaoY1-9D. According to the latest ICTV classification criteria, GaoY1-9D is a novel phage and belongs to Drexlerviridae.
Nucleotide sequence accession number The GenBank accession number for Escherichia phage GaoY1-9D is PP971806.