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.
Table 1 The K.peumoniae used for host range determination
Strain
|
Region of isolation
|
Infection by
Phage Bp5
|
Strain
|
Region of isolation
|
Infection by Phage Bp5
|
GX L15
|
NanNing,GuangXi
|
-
|
GX 20-1
|
NanNing,GuangXi
|
-
|
GX L40
|
NanNing,GuangXi
|
-
|
GX RS3
|
NanNing,GuangXi
|
-
|
GX L44-1
|
NanNing,GuangXi
|
-
|
GX L22
|
NanNing,GuangXi
|
-
|
GX L27
|
NanNing,GuangXi
|
-
|
GX L59
|
NanNing,GuangXi
|
-
|
GX L13
|
NanNing,GuangXi
|
-
|
CX L30
|
NanNing,GuangXi
|
-
|
GX SL39
|
NanNing,GuangXi
|
-
|
GX L3-1
|
NanNing,GuangXi
|
-
|
GX 08
|
NanNing,GuangXi
|
-
|
GX 21
|
NanNing,GuangXi
|
-
|
GX L63
|
NanNing,GuangXi
|
+
|
GX L68
|
NanNing,GuangXi
|
-
|
GX SL26
|
NanNing,GuangXi
|
-
|
GX L48
|
NanNing,GuangXi
|
-
|
GX L18
|
NanNing,GuangXi
|
-
|
GX L67
|
NanNing,GuangXi
|
-
|
GX L28
|
NanNing,GuangXi
|
+
|
GX L55
|
NanNing,GuangXi
|
-
|
GX 13
|
NanNing,GuangXi
|
-
|
GX L34
|
NanNing,GuangXi
|
-
|
GX 49
|
NanNing,GuangXi
|
-
|
GX L11
|
NanNing,GuangXi
|
-
|
GX L01
|
NanNing,GuangXi
|
-
|
GX L03
|
NanNing,GuangXi
|
-
|
GX 06-1
|
NanNing,GuangXi
|
-
|
GX L45-1
|
NanNing,GuangXi
|
-
|
GX L56
|
NanNing,GuangXi
|
-
|
GX L02
|
NanNing,GuangXi
|
-
|
GX 20-2
|
NanNing,GuangXi
|
-
|
GX 54-2
|
NanNing,GuangXi
|
-
|
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 (OD600) 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 (OD600<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 (MLD100) of K pneumoniae was detected as 4.0 ×107cfu/ 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%.
Genome overview
BLASTn analysis showed that vB_KpnM _Bp5 shares 94.06% identity (94% genome coverage) with a K pneumoniae phage vB_KpnP_SU552A of complete genome (KP708986.1) identify. The RAST annotation results indicated that the genome contained only 52 open reading frames (ORFs), and that ATG was used as the start codon for all 52 ORFs. Based on the homology comparisons, 52 ORFs were assigned significant similarity (E value B 1E-4; Table2) to other proteins in the GenBank database. The nucleic acid coding sequences ranged in length from 153 to 3699 bp, corresponding to protein sequence lengths of 51–1232 aa. Together, the ORFs accounted for a total of 40473 bp, with a gene density as high as 92.25%.
|
|
Table 2 ORF analysis of the vB_KpnM _Bp5 genome
|
|
|
|
|
Identity
|
ORF
|
Hit_name
|
Hit_description
|
length
|
Identity
|
value
|
|
1
|
YP_009204790.1
|
hypothetical protein SU552A_01 [Klebsiella phage vB_KpnP_SU552A]
|
71
|
98.59%
|
1.50E-31
|
|
2
|
YP_009188313.1
|
hypothetical protein [Klebsiella phage Kp2]
|
190
|
95.79%
|
3.10E-95
|
|
3
|
YP_009199887.1
|
hypothetical protein SU503_03 [Klebsiella phage vB_KpnP_SU503]
|
74
|
85.14%
|
8.30E-36
|
|
4
|
YP_009302709.1
|
hypothetical protein kpv71_05 [Klebsiella phage KpV71]
|
77
|
76.62%
|
3.50E-29
|
|
5
|
YP_009188747.1
|
hypothetical protein kpv41_05 [Klebsiella phage KpV41]
|
87
|
97.33%
|
1.70E-36
|
|
6
|
YP_009006027.1
|
hypothetical protein F19_07 [Klebsiella phage F19]
|
59
|
94.92%
|
8.10E-26
|
|
7
|
YP_003347658.1
|
hypothetical protein [Klebsiella phage KP34]
|
69
|
95.65%
|
5.60E-34
|
|
8
|
APZ82721.1
|
hypothetical protein kpv74_09 [Klebsiella phage vB_KpnP_KpV74]
|
655
|
88.71%
|
0.00E+00
|
|
9
|
APZ82722.1
|
putative peptidase [Klebsiella phage vB_KpnP_KpV74]
|
348
|
97.70%
|
2.30E-205
|
|
10
|
AIT13582.1
|
hypothetical protein BO1E_0013 [Klebsiella phage phiBO1E]
|
154
|
53.25%
|
2.90E-35
|
|
11
|
No hits
|
|
|
|
|
|
12
|
YP_009188325.1
|
DNA primase/helicase [Klebsiella phage Kp2]
|
261
|
98.08%
|
2.10E-145
|
|
13
|
YP_009204805.1
|
putative DNA helicase [Klebsiella phage vB_KpnP_SU552A]
|
426
|
99.52%
|
9.20E-241
|
|
14
|
YP_009188759.1
|
hypothetical protein kpv41_17 [Klebsiella phage KpV41]
|
51
|
98.04%
|
5.20E-21
|
|
15
|
YP_009204807.1
|
putative DNA polymerase [Klebsiella phage vB_KpnP_SU552A]
|
796
|
98.99%
|
0.00E+00
|
|
16
|
YP_009199903.1
|
hypothetical protein SU503_19 [Klebsiella phage vB_KpnP_SU503]
|
182
|
88.46%
|
9.90E-91
|
|
17
|
YP_009188331.1
|
hypothetical protein [Klebsiella phage Kp2]
|
73
|
98.46%
|
4.40E-33
|
|
18
|
YP_009204809.1
|
hypothetical protein SU552A_20 [Klebsiella phage vB_KpnP_SU552A]
|
344
|
98.84%
|
1.10E-202
|
|
19
|
APZ82733.1
|
hypothetical protein kpv74_21 [Klebsiella phage vB_KpnP_KpV74]
|
77
|
95.31%
|
5.80E-25
|
|
20
|
YP_009204811.1
|
large tegument protein [Klebsiella phage vB_KpnP_SU552A]
|
277
|
97.47%
|
8.60E-145
|
|
21
|
YP_009188766.1
|
hypothetical protein kpv41_24 [Klebsiella phage KpV41]
|
84
|
97.62%
|
5.50E-36
|
|
22
|
YP_009280692.1
|
hypothetical protein kpv475_23 [Klebsiella phage KpV475]
|
92
|
96.74%
|
1.10E-45
|
|
23
|
YP_009188768.1
|
hypothetical protein kpv41_26 [Klebsiella phage KpV41]
|
123
|
97.54%
|
3.60E-60
|
|
24
|
APZ82738.1
|
hypothetical protein kpv74_26 [Klebsiella phage vB_KpnP_KpV74]
|
53
|
96.23%
|
6.90E-16
|
|
25
|
YP_009204817.1
|
hypothetical protein SU552A_28 [Klebsiella phage vB_KpnP_SU552A]
|
53
|
100.00%
|
7.40E-23
|
|
26
|
YP_009204818.1
|
putative 5'-3' exonuclease [Klebsiella phage vB_KpnP_SU552A]
|
322
|
99.69%
|
2.10E-192
|
|
27
|
APZ82743.1
|
putative HNH endonuclease [Klebsiella phage vB_KpnP_KpV74]
|
153
|
58.73%
|
2.60E-35
|
|
28
|
YP_009098366.1
|
putative endonuclease [Klebsiella phage NTUH-K2044-K1-1]
|
140
|
82.31%
|
2.20E-55
|
|
29
|
YP_009204821.1
|
putative polynucleotide kinase/phosphatase [Klebsiella phage vB_KpnP_SU552A]
|
164
|
98.78%
|
4.40E-90
|
|
30
|
YP_009204822.1
|
hypothetical protein SU552A_33 [Klebsiella phage vB_KpnP_SU552A]
|
104
|
100.00%
|
4.30E-54
|
|
31
|
AOT23867.1
|
DNA-dependent RNA polymerase [Klebsiella phage KP-Rio/2015]
|
822
|
98.18%
|
0.00E+00
|
|
32
|
AIT13609.1
|
hypothetical protein BO1E_0040 [Klebsiella phage phiBO1E]
|
146
|
99.32%
|
2.00E-78
|
|
33
|
YP_003347631.1
|
hypothetical protein [Klebsiella phage KP34]
|
87
|
100.00%
|
1.30E-35
|
|
34
|
YP_009204826.1
|
head-tail connector protein [Klebsiella phage vB_KpnP_SU552A]
|
531
|
98.87%
|
9.10E-294
|
|
35
|
YP_009204827.1
|
putative scaffolding protein [Klebsiella phage vB_KpnP_SU552A]
|
280
|
99.64%
|
4.50E-151
|
|
36
|
AOT23872.1
|
capsid protein [Klebsiella phage KP-Rio/2015]
|
339
|
97.05%
|
4.10E-191
|
|
37
|
YP_009280706.1
|
hypothetical protein kpv475_37 [Klebsiella phage KpV475]
|
60
|
96.67%
|
4.00E-20
|
|
38
|
YP_009280707.1
|
putative tail tubular protein A [Klebsiella phage KpV475]
|
186
|
99.46%
|
4.90E-101
|
|
39
|
YP_009188784.1
|
putative tail tubular protein B [Klebsiella phage KpV41]
|
786
|
98.98%
|
0.00E+00
|
|
40
|
YP_009204832.1
|
putative internal virion protein B [Klebsiella phage vB_KpnP_SU552A]
|
195
|
97.95%
|
1.60E-94
|
|
41
|
YP_009188357.1
|
hypothetical protein [Klebsiella phage Kp2]
|
894
|
99.11%
|
0.00E+00
|
|
42
|
YP_009199928.1
|
putative internal core protein [Klebsiella phage vB_KpnP_SU503]
|
1232
|
98.62%
|
0.00E+00
|
|
43
|
APZ82760.1
|
putative tail fiber protein [Klebsiella phage vB_KpnP_KpV74]
|
602
|
90.00%
|
2.40E-67
|
|
44
|
No hits
|
|
|
|
|
|
45
|
YP_009199930.1
|
putative DNA maturase A [Klebsiella phage vB_KpnP_SU503]
|
100
|
94.12%
|
8.00E-35
|
|
46
|
YP_009188791.1
|
putative DNA maturase B [Klebsiella phage KpV41]
|
618
|
99.19%
|
0.00E+00
|
|
47
|
YP_003347646.1
|
hypothetical protein [Klebsiella phage KP34]
|
124
|
98.37%
|
3.60E-60
|
|
48
|
AOT23882.1
|
hypothetical protein KPRIO2015_44 [Klebsiella phage KP-Rio/2015]
|
60
|
96.67%
|
6.10E-21
|
|
49
|
YP_003347648.1
|
hypothetical protein [Klebsiella phage KP34]
|
134
|
99.25%
|
5.50E-62
|
|
50
|
YP_009006072.1
|
Putative holin protein F19_49 [Klebsiella phage vB_KpnP_SU552A]
|
83
|
98.00%
|
8.00E-111
|
|
51
|
YP_009204842.1
|
putative endolysin [Klebsiella phage vB_KpnP_SU552A]
|
202
|
95.05%
|
1.50E-99
|
|
52
|
YP_009098386.1
|
putative HNH endonuclease [Klebsiella phage NTUH-K2044-K1-1]
|
153
|
94.74%
|
6.80E-85
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Note: “No hits” indicates no significant hits detected for a particular amino acid sequence
Functional ORF Analysis
Analysis of protein sequences using BLASTp and RAST revealed that Structural protein ORF20 and ORF36, with a length of 299 and 377 bp, respectively, were the major tegument proteins. While ORF34 appeared to be phage head-tail connector proteins. In addition, ORF35, as a scaffold protein, had a supporting and protective effect on the overall structure of phage. ORF38-39 were associated with Tail tubular protein A (TTPA), these were structural tail proteins of K pneumoniae bacteriophage. The gene clusters encoding these proteins played a significant role in phage infection and adsorption of host bacteria. Specifically, seven proteins were involved in the replication and regulation of phage DNA. ORF12-13, were likely to be a DNA primase/helicase protein. ORF 15 encoded DNA polymerase protein with a length of 796 bp was detected. Meanwhile, ORF 26-28 had the functional characteristics of endonuclease, played a vital role in Phage DNA replication regulation. ORF31 showed the greatest identity to phage DNA-dependent RNA polymeras proteins. As the phage genome was small, no relevant genes coding for DNA topoisomerases, or transcriptional regulators were identified. ORF50 was shown to be a holin, a protein that perforates the bacterial cell membrane, while ORF51 (endolysin of phage) was associated with phage cleavage. Therefore, phage vB_KpnM _Bp5 might mediate host cell lysis by simultaneously expressing both proteins. A detailed description of the ORFs and their identified functionality was shown in Fig 4.
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.
Table 3 vB_KpnM _Bp5 genome alignment result using BLAST program
Accesssion
|
Description
|
Max.score
|
Total score
|
E.value
|
Query Cover (%)
|
Identity (%)
|
KP708986.1
|
Klebsiella phage vB_KpnP_SU552A, complete genome
|
27855
|
56643
|
0.0
|
86
|
94.06
|
KT964103.1
|
Klebsiella phage vB_KpnP_KpV41, complete genome
|
25531
|
52717
|
0.0
|
82
|
92.99
|
GQ413938.2
|
Klebsiella phage KP34, complete genome
|
25130
|
48598
|
0.0
|
82
|
92.31
|
KM576124.1
|
Klebsiella phage phiBO1E, complete genome
|
24640
|
48990
|
0.0
|
84
|
90.91
|
KX856662.1
|
Klebsiella phage KP-Rio/2015, complete genome
|
24221
|
50772
|
0.0
|
83
|
91.70
|
KX211991.1
|
Klebsiella phage vB_KpnP_KpV475, complete genome
|
23363
|
48647
|
0.0
|
81
|
90.81
|
KU666550.1
|
Klebsiella phage vB_KpnP_KpV71, complete genome
|
22692
|
54214
|
0.0
|
84
|
94.17
|
KX587949.2
|
Klebsiella phage phiKpS2,
complete genome
|
19867
|
46571
|
0.0
|
82
|
90.84
|
KP708985.1
|
Klebsiella phage vB_KpnP_SU503, complete genome
|
13538
|
49058
|
0.0
|
83
|
93.78
|
KT367886.1
|
Klebsiella phage vB_Kp2, complete genome
|
13381
|
38967
|
0.0
|
75
|
87.63
|