Characterization and genome analysis of novel phage vB_KpnM _Bp5 infecting Klebsiella pneumonia CURRENT POSTED

Background With the incidence of antibiotic resistance reaching crisis point, it is imperative to find alternative treatments for multidrug-resistant infections. Using phage for pathogen control might be a promising treatment option to combat bacterial resistance. Results In this study, a lytic phage, designated vB_KpnM _Bp5, was isolated from pig faecal sample in Nanning, Guangxi province of China, and classified as a member of the family Muscle virus based on electron microscopy analysis. A one-step growth curve of the phage at the optimal MOI revealed that the latent time was 40 min and the burst size was 24 PFU/cell, indicative of good lysis capacity. Whole genome sequencing showed that phage vB_KpnM _Bp5 had a small dsDNA genome of 43872 bp. BLASTn analysis showed that it shared 94.06% identity (94% genome coverage) with Klebsiella phage vB_KpnP_SU552A of complete genome idefix. RAST genome analysis showed that the phage had 50 ORFs due to its small genome size, and the number of functional proteins was consistent with other phages. To evaluate the therapeutic effect of Klebsiella pneumoniae infection in mice, the results showed that phages provided vB_KpnM _Bp5better protection. Conclusion The phage vB_KpnM _Bp5 had the characteristics of broad host spectrum, strong environmental adaptability, short incubation period, large outbreak amount, and can cure the mouse model infected by Klebsiella pneumoniae. These findings suggested that phage vB_KpnM _Bp5 could be considered a potential therapeutic or prophylactic candidate against Klebsiella pneumonia infection.


Abstract
Background With the incidence of antibiotic resistance reaching crisis point, it is imperative to find alternative treatments for multidrug-resistant infections. Using phage for pathogen control might be a promising treatment option to combat bacterial resistance. Results In this study, a lytic phage, designated vB_KpnM _Bp5, was isolated from pig faecal sample in Nanning, Guangxi province of China, and classified as a member of the family Muscle virus based on electron microscopy analysis. A one-step growth curve of the phage at the optimal MOI revealed that the latent time was 40 min and the burst size was 24 PFU/cell, indicative of good lysis capacity. Whole genome sequencing showed that phage vB_KpnM _Bp5 had a small dsDNA genome of 43872 bp. BLASTn analysis showed that it shared 94.06% identity (94% genome coverage) with Klebsiella phage vB_KpnP_SU552A of complete genome idefix. RAST genome analysis showed that the phage had 50 ORFs due to its small genome size, and the number of functional proteins was consistent with other phages. To evaluate the therapeutic effect of Klebsiella pneumoniae infection in mice, the results showed that phages provided vB_KpnM _Bp5better protection. Conclusion The phage vB_KpnM _Bp5 had the characteristics of broad host spectrum, strong environmental adaptability, short incubation period, large outbreak amount, and can cure the mouse model infected by Klebsiella pneumoniae.
These findings suggested that phage vB_KpnM _Bp5 could be considered a potential therapeutic or prophylactic candidate against Klebsiella pneumonia infection.

Background
Klebsiella pneumoniae is generally considered as a pathogen with the propensity for acquiring antibiotic resistance [1]and could cause lots of infections including pneumonia, urinary tract infections, sepsis, and soft tissue infections. K pneumoniae is widely spread in farming environments, especially livestock and poultry farms. It is also a significant nosocomial pathogen due to its resistance to antibiotics [2,3]. In addition, as a result of increasing international mobility, antibiotic-resistant K pneumoniae tends to disseminate rapidly through different countries as well as evolves into real pandemics [4]. K pneumoniae has spread rapidly and becomes endemic in several countries (e.g. USA [5], China, Israel [6], Italy [7] and Colombia [8][9][10],Portugal [11],Greece [12]). It is now a major cause of healthcare-associated infections correlated with high morbidity and mortality rates [10]. Relevant study has shown that pathogenic bacteria developed resistance much faster than the development of new antibiotics [13].
Using phage for pathogen control is an alternative treatment strategy to fight against bacterial antibiotic resistance [14]. Phages are natural viruses that infect bacteria, and exist as the most abundant biological entities in the biosphere [15]. Phages only kill their corresponding host bacteria and rely on host bacteria for their reproduction. Bacterial antibiotic resistance has no effects to impede phage therapy on bacterial infections.
Hence phage plays an important role in the application of antimicrobial pathogens in the fight.
The host range of a bacteriophage is usually narrow. In addition, phage-resistant mutant bacteria arise almost instantly upon exposure to a phage, which severely limits the therapeutic use of phage on bacterial infections. To overcome this hurdle, a cocktail consisting of several different phages is frequently used to reduce the evolution of resistant bacteria and to maintain highly lytic efficacy [16]. Accordingly, it is necessary to isolate and characterize new phages. In the current study, we isolated lytic phage vB_KpnM _Bp5, which showed activity against K pneumoniae, from farm sewage and carried out genome sequencing and analysis of its biological characteristics.

Phage isolation and morphology
To isolate bacteriophages with specific lytic activity against K pneumoniae, various environmental samples were collected from sewage taken from pig farms. A new lytic bacteriophage, named as vB_KpnM _Bp5, was isolated. The phage plaque was clean and bright, with neat edge and no halo ring, which showed typical lytic phage characteristic (Fig 1a). Electron microscopy showed that the phage tubular tail was approximately 56 nm in length, and the head was 53 nm in diameter (Fig. 1b). According to the provisions of the International Classification Committee of Viruses, the phage was assigned to the family Caudovirales, Myoviridae.

Host specificity
To test whether vB_KpnM _Bp5 could lyse human K.peumoniae isolates, the host range of phage vB_KpnM _Bp5 was examined on 34 K pneumoniae strains isolated from fetal swabs and urine collected in a hospital in Nanning, Guangxi Zhuang autonomous region. The result showed that vB_KpnM _Bp5 exhibited an ability to produce plaques in 2 human isolates, respectively, K pneumoniae strains GX L63 and GX L28. (Table 1). It indicated that phage Bp5 had a host range of both pig and human isolates.

One-step growth
An one-step growth experiment was conducted to determine the latent time period and burst size of phage vB_KpnM _Bp5 (Fig.2a).The assays revealed a latent period, defined as the time interval between the adsorption and the beginning of the first burst,the results showed that the incubation period was about 5min, and the outbreak period was about 40min. 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 was determined to be 24 plaque-forming units (PFU)/cell for phage vB_KpnM _Bp5.

Thermal and pH stability
When heated to 50 °C, The phage titer of vB_KpnM _Bp5 decreased rapidly with the increase of water temperature (Fig.2b). When water bath temperature reached 80 ℃, no living phages were detected, which showed high temperature affected the activity of phages. According to the result, phages could maintain the stable activity when temperature heated to 30 ~ 50 ℃. In addition, Phage vB_KpnM _Bp5 exhibited stable activity at pH 4.0-10.0 (Fig.2c).Moreover, phage titer declined sharply with the enhancement of acid or alkalinity.

Bacteriolytic activity
The bacteriolytic activity of phage vB_KpnM _Bp5 was tested on an early exponential phase culture of K pneumoniae (Fig.2d). The absorbance (OD 600 ) of the phage-infected culture dropped rapidly compared with the uninfected control from 1 to 5 h post infection.
Although the bactericidal effect exhibited slight differences with changes in the MOI (multiplicity of infection), both 1 MOI and 0.001 MOI phage-infected culture showed significantly (p<0.01) inhibited bacterial growth after 2 h. The result showed using MOI=1 phage to infect culture, the absorbance decreased rapidly and remained at a very low level (OD 600 <0.1), while using MOI=0,001 phage to infect, the absorbance first raised then descended. It indicated that the phage bacteriolytic activity would increase with the rising of its concentration.

Evaluation of phage therapy
To evaluate the effect of phage therapy on K.pneumoniae infected mice, the minimum lethal dose (MLD 100 ) of K pneumoniae was detected as 4.0 ×10 7 cfu/ piece. Each mice were infected with K.pneumoniae at the MLD. At the same time, phage had a good therapeutic effect, the advance treatment group and the current treatment group showed that the protection rate of phage on mice was up to 100.00%. Meanwhile, the protection rate of phage on mice in the delayed treatment group was only 60.00%. All mice in the bacterial infection group died, and the mice in the control group did not die (Fig.3).
Phage vB_KpnM _Bp5 complete genome sequencing was conducted using next-generation sequencing (NGS). A total of about 5,304,348 reads were detected and 99.33% of reads were high quality reads. The genome was assembled using A5-miseq v20150522 and SPAdesv3.9.0 assembler. To be specific, the complete genome length of vB_KpnM _Bp5 was 43872bp, and 94.93% of the whole reads were matched onto the complete genome.
The distribution of each base was the same as, the average GC content was 53.90%.

Evolutionary Analysis
Genome BLAST results illustrated that the vB_KpnM _Bp5 genome showed very high similarity with other current NCBI published genomes in the aspects of complete genome (Table 3). To illustrate the evolutionary relationship between vB_KpnM _Bp5 and all of the other known K pneumoniae phage representative strains, their multiple alignments were performed based on their complete genomes (Fig.5). The Genetic evolution tree showed that vB_KpnM _Bp5 was on the same branch as K pneumonia phage vB_KpnP_SU552A, and closely related to the K pneumoniae phage genome, such as vB_KpnP_KpV41 KP -Rio_2015 myPSH1235 et al.

Discussion
With the advent of molecular biology, we were now better able to understand the coevolved relationships between phage and their hosts, which could facilitate individualized treatments by using phages for bacterial infection [17].These advent of molecular biology gave us greater insights on how to most effectively use bacteriophage as potential therapeutic agents. Therefore, the purpose of this study was to analyze the biological characteristics and complete genome of phage, so as to provide valuable knowledge and insight for single phage or cocktail therapy.
Recently, a patient with cystic fibrosis with a disseminated Mycobacterium abscessus infection was treated with a three-phage cocktail following bilateral lung transplantation, and achieve good therapeutic results [18][19][20]. Because the need to control MDR infections was urgent, phages were being newly studied as potential antibiotic alternatives. Phages were able to perform the function of phagocytic bacteria, mainly because it could undergo two different life cycles: the holing cycle and the endolysin cycle [21]. In this study, ORF 50 and ORF 51 were defined as regions encoding holin and endolysin .Phage endolysins accumulated in the host cell cytoplasm during phage development [22], until holins suddenly induce formation of holes in the cytoplasmic membrane [23].These holes provided a pathway for endolysin release to the cell wall that was rapidly cleaved, leading to cell burst [24].Therefore, the holin function had the crucial role of defining the time of lysis, which was fundamental for phage fitness. By clearly localized the enzymes of holin and lyase genes, it could provide a feasible idea for exploring the phage lysis mechanism.
Meanwhile, further exploration was required to improve the application of phage vB_KpnM _Bp5 as antibacterial agents in animals. For example, increased phage concentration might improve the therapeutic effect of the treatment, a cocktail including single phage and several other potential therapeutic phages might broad the lytic spectrum and reduce the possibility of phage resistance. Overall, the study of phages for using in antibacterial therapies was still in its infancy, meant that there was much room to explore alternative approaches and develop novel phage-based therapies. In summary, our study on the vB_KpnM _Bp5 provided new information, which from biocharacteristic analysis and whole genome sequencing. It could provide the basis of developing phage therapy of K pneumoniain fections.

Conclusion
The phage vB_KpnM _Bp5 had the characteristics of broad host spectrum, strong environmental adaptability, short incubation period, large outbreak amount, and could cure the mouse model infected by K pneumoniae. These findings suggested that phage vB_KpnM _Bp5 could be considered a potential therapeutic or prophylactic candidate against K pneumonia infection.

Methods
K. peumoniae strain J5 was isolated from farm sewage. A total of 34 K. pneumoniae strains were isolated from stool swabs and urine collected from a hospital in Nanning, Guangxi province. All bacterial strains were routinely cultivated at 37°C using lysogeny broth (LB).

Phage isolation and purification
The conventional double-layered agar method was used to isolate phages and enumerate plaque forming units (PFUs) [25]. K. pneumoniae was routinely used as the host bacterial strain for phage isolation. Plaque morphologies were observed after 12 h -post-incubation.
Bacterial cultures in the exponential phase were inoculated with sewage effluent collected from a pig farm in Nanning, Guangxi, China. The mixture were initially incubated for 12-16 h at 37 ℃. Then it was centrifuged for 10 min at 12000 rpm, and filtered through a 0.25 μm pore size membrane filter to become the original liquid containing phage. The filtered phage lysate will be concentrated for 10 times dilution and join the corresponding host bacterium, using the method of double layer tablets after observing plaque growth situation at 37 ℃ for 12 h. Uniform size plaque was screened and titer was determined.
Phages were stored at 4℃ for further experiments.

Electron microcopy
The purified phage sample was loaded onto a copper grid followed by negative staining with 2 % Phosphotungstic acid (PTA, 2% w/v) staining and drying. Phage sizes were calculated by means of at least 3 measurements.

Host range analysis
The host range of the phage was determined by spot test [26]. Lysis zone or plaque formation was monitored upon application of 10 μl of phage lysate, adjusted to contain 1>10 7 PFU. ml − 1 , by spotting assay.

One-step growth
One-step growth experiments were carried out by a modification of methods described elsewhere [27,28].The logarithmic phase mixed with an overdose of phage host (MOI 10), 37 ℃ warm bath after 15 min 12000 rpm centrifugal 1 min, discard supernatant and wash with LB to precipitate .Then, suspended in LB that had been pre-warmed to 37 °C, followed by incubation at 37 °C. Samples were taken at 10 min intervals (up to 90min) and immediately diluted, and phage titers were then determined by double-layered agar plate method.

Thermal and pH stability
For thermos-stability testing, samples of the isolated phage were incubated at 30, 40, 50, 60, 70, 80℃, and aliquots were taken after 30 and 60 min to be titer by the double-layer agar method. For pH stability testing, samples of the isolated phage were mixed in a series of tubes containing SM of different pH values (2.0-11.0, adjusted using NaOH or HCl), incubated for 2 h at 37 ℃, and then titer by the double-layer agar plate method [29,14].

Bacteriolytic activity
The host bacteria were cultured overnight and diluted to 1× 10 8  University. According to the standards committee, the survival mice were euthanized by cervical dislocation.

Assess the virulence of host bacteria
To evaluate the virulence of host bacteria in vivo with the minimum lethal dose, that was, The survival mice were euthanized by cervical dislocation.

Bacteriophage genome extraction
Phage gDNA was isolated from purified phages (procedure described above) and extracted

Competing Interests
The authors declare that they have no conflict of interest.

Availability of data and materials
The data were presented in the main manuscript and available to readers.   Genome map of vB_KpnM _Bp5, the linear genome of vB_KpnM _Bp5 is depicted in a circularized format. Comparative analysis of these two phages was conducted using Easyfig, with nucleotide identity above 90%. Well conserved segments are paired by shaded regions,and arrows indicate the direction of transcription for the predicted ORFs.

Supplementary Files
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