Characterization and genome analysis of a novel phage Kayfunavirus TM1

Escherichia coli is a common conditional pathogen, for which antibiotic therapy is considered an effective treatment. The imprudent use of antibiotics has led to the increase of multiple-antibiotic-resistant E. coli species. With the incidence of antibiotic resistance reaching a crisis point, it is imperative to find alternative treatments for multidrug-resistant infections. Using phage for pathogen control is a promising treatment option to combat bacterial resistance. In this study, a novel virulent Podoviridae phage Kayfunavirus TM1 infecting Escherichia coli was isolated from pig farm sewage in Guangxi, China. The one-step growth curve with the optimal multiplicity of infection of 0.01 revealed a latent period of 10 min and a burst size of 50 plaque-forming units per cell. The stability test reveals that it is stable from 4 to 60 °C and pH from 3 to 11. The double-stranded DNA genome of phage Kayfunavirus TM1 is composed of 39,948 base pairs with a GC content of 50.03%.


Introduction
Along with the rapid increase in pork production, both the number and the size of intensive swine farms have grown [1]. The occurrence of bacterial diseases seriously restricts the development of the swine industry, especially diseases caused by Escherichia coli (E. coli). Escherichia coli is the most common cause of Gram-negative breeding industry infections [2]. Post weaning diarrhea (PWD) due to E.coli is an economically important disease in pig production worldwide, affecting pigs during the first 2 weeks after weaning and is characterized by sudden death or diarrhea, dehydration, and growth retardation in surviving piglets, which brings great harm to animal husbandry [3]. Because of the long-term excessive use of antibiotics in the pig industry, the antibiotic resistance of Gram-negative bacteria has rapidly increased. Domestic pigs have become an extensive reservoir of antibiotic-resistant bacterial strains and genes, which can seriously endanger public health [4]. At the same time, multidrug-resistant E. coli strains are becoming increasingly common, presenting challenges for clinical treatment. In Mingyang Li's results, E.coli isolates showed various resistances to five commonly used antibiotics (ciprofloxacin, gentamicin, tetracycline, ampicillin, and florfenicol) and they had the highest resistance rate to tetracycline [5]. Furthermore, the development of antibiotics resistance is far faster than new antibiotic development, and as the largest producer and user of antibiotics globally, China banned all antibiotic growth promoters (AGPs) on January 1, 2020 [6]. So we need a global effort to find the potential solutions with higher public health significance to replace antibiotics. Such as natural products, new antibiotics, bacteriophages.
Bacteriophages are bacterial viruses that can infect and lyse bacterial cells. The phages are widely distributed in nature that can be easily isolated from soil, sewage, and feces [7]. There are around 10 7 phages per ml in liquid environments (e.g. seawater) and up to 10 9 per ml in sediments Edited by Andrew Millard. [8]. In addition,phages are an inherent element of the human microbiome, and therefore they are well tolerated when used in phage therapy [8]. Phage therapy started with its first clinical application in 1919 [9].The bacteriophages have been studied and used to control bacterial infections of patients in Poland, Georgia, and Russia for nearly a century [10], and can significantly reduce the concentrations of targeted bacteria in pigs [11].
In this study, a new phage Kayfunavirus TM1 was isolated from the sewage of a swine farm in Guangxi, and its biological characteristics, whole-genome sequencing, and evolutionary analysis provided a reliable basis for further research and application of E. coli phage.

Bacterial strains and culture conditions
GXEC-TM1 was the host bacterium of the phage Kayfunavirus TM1 and it was isolated from feces specimens collected from pig at the farm in Guangxi, China. After sequencing the 16S rRNA gene to confirm the bacterial strains, the strain was stored in the culture collection of our laboratory. GXEC-TM1 were stored at − 80 °C and cultivated at 37 °C with shaking at 220 rpm in Luria-Bertani (LB; hopebiol) liquid medium or plated onto solid LB medium containing 1.5% (w/v) agar and cultured at 37 °C for 16 h with aeration.

Phage isolation and purification
GXEC-TM1 was used as the host bacterium, and the phage Kayfunavirus TM1 was isolated from the pig farm sewage in Guangxi, China. Fifty mL of sewage was centrifuged for 10 min at 5000×g, 3 times. The supernatant was removed and filtered through a 0.22 µm microporous membrane. One hundred mL of filtered effluent was mixed with 4 mL double strength LB and 100 µL 1 × 10 8 CFU/mL GXEC-TM1 [12]. The above combination was cultured at 180×g (37 °C) for 12 h. Then the mixture was centrifuged (8000×g, 10 min) and the supernatant was filtered through a 0.22 µm microporous membrane and stored as the phage stock. The filtered mixture was serially diluted seven times, tenfold each time. 100 µL of phage dilution was mixed with 200µL of GXEC-TM1 (1 × 10 CFU/mL) and incubated at 37 °C for 5 min. Then added the mixture in 5 mL 0.6% LB top agar (50 °C), and the mixture was poured onto the LB plate. The plates of phage were incubated at 37 °C for 12 h to obtain phage plaques. Picked the transparent plaque and dissolved it in phage buffer. The purified phage was obtained by repeating the above procedure five times and the phages were stored at 4 °C for further experiments.

Determination of host range of phage Kayfunavirus TM1
To determine the host range of phage Kayfunavirus TM1, we chosed 22 strains of bacteria isolated from pigs in Guangdong and Guangxi. Added 2 µL purified phage solution on the center of the double-layer agar medium that was covered with different bacteria. The host range was characterized by the spot test and assayed at 37 °C for 12 h. The tested bacteria which could form clear plagues were considered susceptible to phage infection [13].
One-step growth experiments were carried out by a modification of methods described by Zhang C [12]. The host strain (1 × 10 8 CFU/mL) and phage Kayfunavirus TM1 (1 × 10 6 PFU/mL) were mixed at the MOI of 0.01 and incubated at 37 °C for 15 min, then centrifuged the mixture (12,000×g, 1 min) to remove unabsorbed free phage. Added 3 mL LB (37 °C) to the precipitate and incubated at 37 °C. Every 10 min collected 100 µL sample (up to 90 min) and determined the phage titers by the double-layered agar plate method. The calculation formula of burst size was: burst size = phage titer at the end of lysis/number of host strain at the beginning of infection. All tests were repeated three times.

Thermal and pH stability
To determine the thermostability of phage Kayfunavirus TM1, the phage suspensions were incubated at various temperatures (4 °C, 26 °C, 40 °C, 60 °C and 80 °C) for 30 min, 60 min, and 90 min, and then the phage titer was determined. To evaluate the stability of the phage at different pH levels, the purified phages were incubated in SM buffer at different pH levels ranging from 2 to 12 for 120 min. Phage samples were titered using the soft agar overlay method. All tests were repeated three times.

In-vitro antibacterial curve
We determined the effect of phage Kayfunavirus TM1 against GXEC-TM1 at the best MOI by a bacterial growth assay. The host bacteria were cultured overnight and diluted to 1 × 10 8 CFU/mL, then diluted the phage Kayfunavirus TM1 to 1 × 10 6 PFU/mL. Mixed the diluted bacteria and phage. The final mixtures' OD 450 was 0.1 and MOI was 0.01. Two hundred µL of the mixtures were added to the 96-well microplate and the microplate was placed in the multiskan spectrum at 37 °C for 16 h. The test conditions included the wavelength range was 450 nm, the time of sweep spacing was 1 h, and the frequency of oscillation was 180 rpm. All tests were repeated three times.

Nucleic acid extraction and whole-genome sequencing of phage Kayfunavirus TM1
Phage DNA was isolated from purified phages and extracted by an alkaline lysis method [12]. After the phage particles was suspended in SM buffer, sodium dodecyl sulfate (SDS) (final concentration, 0.5%) and proteinase K (final concentration, 50 μg/mL) were added. DNA/RNA enzyme actioned the mixtures at 37 °C, 30 min. The mixture was vortexed thoroughly and incubated at 56 °C for 16 h. An equal volume of phenol-chloroform (1:1) was added to remove the proteinaceous material. The extraction was repeated twice, and the DNA was precipitated according to ethanol precipitation procedures. The phage particles was dissolved in 30 μL of distilled water, and the nucleic acids were separated using 0.8% agarose gel electrophoresis, stained with ethidium bromide, analyzed the purity and concentration of the phage nucleic acid by observing the number and brightness of the bands under ultraviolet rays (UV) light. After digestion, electrophoresis of the samples in 0.8% agarose containing ethidium bromide (1 μg/mL) was performed. The method of sequencing adopted the whole genome shotgun (WGS).

Full genome analysis of phage Kayfunavirus TM1
Sequencing library construction, sequencing and data quality control were performed by Personalbio (Shanghai Personal Biotechnology Co., Ltd, China). The sequencing platform was Illumina Miseq, sequencing mode was pairedend (2 × 250 bp) and library insert size was 400 bp. The tools of genome visualization chosed The CGView Server (CGView Server) and Easyfig_2.2.5_win. Easyfig is creating linear comparison figures of multiple genomic loci based on BLAST [12]. The whole-genome sequence was annotated using Subsystem Technology (https:// rast. nmpdr. org/ rast. cgi). We also carried out BLASTp (https:// blast. ncbi. nlm. nih. gov/ Blast. cgi) for further verification of the predicted proteins. The phylogenetic tree of the phage Kayfunavirus TM1 was constructed based on the large terminator subunit by MEGA-X [14].

Statistical analysis
All experiments have been performed 3 times. All statistical analyses were performed using GraphPad Prism 8 (Graph-Pad Software, Inc., San Diego, CA, USA). The significance of experimental data was determined with multiple tests. The error bars represent the standard deviation (SD) of the mean.

Nucleotide sequence accession number
The nucleotide sequence reported in the present study was listed in the GenBank nucleotide sequence database under the accession number OM908436.1.

Phage morphology
Phage Kayfunavirus TM1 was isolated from pig farm sewage in Guangxi, China. Phage Kayfunavirus TM1 formed small, clear, and round plaques approximately 2 mm in diameter after incubating on soft agar plates with host bacteria for 5-7 h at 37 °C (Fig. 1A). Electron microscopy showed that the phage tubular tail was approximately 36 nm in length, and the head was 54 nm in diameter (Fig. 1B). In accordance with the International Classification Committee of Viruses, the phage belonged to the order Caudovirales, family Autographiviridae, subfamily Studiervirinae, genus Kayfunavirus according to its morphology, named as Kayfunavirus TM1.

Host range of phage Kayfunavirus TM1
As shown in Table 1, twenty-two strains were tested. Host range analysis revealed that phage Kayfunavirus TM1 had a very high specificity and only cleaved GXEC-TM1, and showed no activity against any of the other strains examined in this study.

Optimal MOI and one-step growth assays
Phage Kayfunavirus TM1 was mixed with the host bacteria in a certain ratio, When the MOI was 0.01, the phage Kayfunavirus TM1 produced the maximum progeny virus ( Fig. 2A). which indicated that 0.01 is the optimal MOI of phage Kayfunavirus TM1. The one-step growth experiment was conducted to determine the latent time period and burst size of phage Kayfunavirus TM1 (Fig. 2B). The results showed that the latent period was 5 min and the outbreak period was 40 min. The burst size was calculated as the ratio of the final number of free phage particles to the number of infected bacterial cells during the latent period, and the burst size of phage Kayfunavirus TM1 was determined to be 20 plaque-forming units PFU/ cell.

The stability analysis of phage Kayfunavirus TM1
The stability of the phage Kayfunavirus TM1 was shown in Fig. 2C and D. For pH stability, the results showed optimal stability of Kayfunavirus TM1 at pH 10, the phage activity stayed at high levels after 2 h of incubation at pH 3-11.  No viable phage was detected at pH 2 and 12 (Fig. 2C). For thermostability, temperatures ranging from 4 to 60 °C won't affect phage vitality, however, temperatures higher than 60 °C would cause vitality decrease. Almost all phage was inactivated at temperatures higher than 80 °C (Fig. 2D).

Antibacterial curve in vitro of phage Kayfunavirus TM1
The bacteriolytic effect of phage Kayfunavirus TM1 was tested on early phase cultures of GXEC-TM1 at the best MOI (Fig. 3). The OD 450 value continued to increase when the GXEC-TM1 was not infected by phage Kayfunavirus TM1. When the GXEC-TM1 was infected, its growth was inhibited and after 7 h the OD 450 value started to increase.

Genomic analysis of Kayfunavirus TM1
According to the genome sequencing results, the linear double-stranded DNA of phage Kayfunavirus TM1 was 39,948 bp in length, with a GC content of 50.03%. Based on RAST and BLASTn analysis, the results revealed that the Kayfunavirus phage DY1 (MT808983.1) had the highest query coverage (89%) containing 94.82% identity. The RAST annotation results indicated that the genome contained only 53 coding domain sequences (CDS), and ATG was the start codon for all CDS. However, only 32 CDS were determined to be functional based on gene predictions and annotation of the genome, the rest of CDS' functions remained unknown ( Table 2). The annotated genes mainly involved 4 functional modules, including phage lysis-related genes, phage morphogenesis genes, DNA packaging genes, replication and regulation related genes. As shown in Table 2 and Fig. 4  identity to phage DNA-dependent RNA polymeras proteins. CDS41 (152 bp), CDS12 (150 bp) and CDS14 (64 bp) was associated with phage cleavage (Fig. 5). As shown in Table 2, phage Kayfunavirus TM1 had 12 CDS, which shared a high identity with Escherichia phage LM33_P1 (LT594300.1). However, only 3 CDS had explicit functionality, including DNA packing, phage morphogenesis, and phage DNA replication and regulation. Moreover, phage Kayfunavirus TM1 contained 2 CDS (Rz-like lysis protein, endolysin) required for phage lysis, which were similar to those in Escherichia phage P762, and 1 CDS (Phage holin) that were similar to Cronobacter phage Dev2, indicating that phage Kayfunavirus TM1 may have similar lysis system to that of Escherichia phage P762 (MW876471.1) and Cronobacter phage Dev2 (HG813241.1). The phage Kayfunavirus TM1 also had 4 CDS (CDS15, CDS21, CDS23, CDS26) which showed the greatest identity to phage tail-associated protein. And these proteins played a significant role in phage infection and the adsorption of host bacteria [15].
In addition, there were no antibiotic resistance and virulence genes were detected in the whole genome of the phage Kayfunavirus TM1.

Phylogeny analysis of phage Kayfunavirus TM1
To analyze the relationships between phage Kayfunavirus TM1 and other phages, evolutionary trees were constructed based on the Phage terminase large subunit Gp19 DNA packaging protein (Fig. 6). The tree showed that phage Kayfunavirus TM1 was closely related to the Escherichia phage Mt1B1_P3 genome, and was closely related to the other

Discussion
As many viral ecologists have said, phages are the most abundant and diverse biological entities on the planet. There are about 1031 kinds of phages on the planet [16,17]. However, only 9,725,803 whole-genome sequences of phages could be retrieved from the NCBI database as of 2022 [Search: phage-NLM (nih.gov)], and it was a small fraction of all phages. As more phages were discovered and more phage sequences were added to reference databases, it would help people identify a larger diversity of viral sequences from metagenomes [18]. In this study, the novel phage Kayfunavirus TM1 was isolated from the sewage of a pig farm in Guangxi, which is significant for the extension of the Escherichia coli phage library. Phage Kayfunavirus TM1 has optimal MOI in 0.01, latent period is 5 min and the outbreak period is 40 min, and a 20 PFU per cell burst size. Phage Kayfunavirus TM1 showed activity from 4 to 60 °C and pH 3-11. It is worth noting that the Phage Kayfunavirus TM1 has a high tolerance to strong bases, especially at pH 11 compared with phage pSb-1, phage P762 [19,20]. In addition, one of the main advantages of bacteriophages is their specificity to infect a few strains only within a species and host-specific phage have previously been used in the treatment of E. coli infections in piglets [21]. In our study, the phage Kayfunavirus TM1 had very high specificity and only cleaved GXEC-TM1. Coincidentally, the phage LM33_P1 was found to exclusively infect O25b strains, and phage LM33_P1 has 12 CDS shared high identity with the phage Kayfunavirus TM1 [22]. The CDS23 is one of the 12 CDS and functional prediction revealed that this CDS encodes the phage tail fiber protein which is usually used as a tool for bacterial pathogen recognition [23][24][25]. Thus, we predicted that the CDS23 is strongly related to host specificity. Of course, it is extremely necessary to explore the function of the 9 hypothetical proteins of the 12 CDS. One limitation of our study is the origin and serotype of the strains we tested, further studies will be required to identify additional strains and serotypes.
In this study, we isolated a novel phage Kayfunavirus TM1 from the sewage. Phage Kayfunavirus TM1 with 39,948 bp in length and a GC content of 50.03%, showed a narrow spectrum. Based on the whole genome analysis and phylogenetic analysis, the phage Kayfunavirus TM1 can be assigned to the genus Kayfunavirus. Author contributions KH: Methodology, investigation, validation, formal analysis, writing-original draft. XM: Formal analysis, software, data curation, writing-original draft, visualization. HL: Writingreview and editing, funding acquisition. LL: Formal analysis, writing-review and editing. XW: Conceptualization, supervision, project administration, funding acquisition, writing-review and editing.

Data availability
The complete genome sequence of phage Kayfunavirus TM1 have submitted to GenBank. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations
Conflict of interest All of the authors declare that they have no conflict of interest.
Ethical approval Not applicable.
Consent to participate All individual participants included in the study are consent to participate.

Consent for publication
All findings were agreed to be published.