Introduction
Escherichia coli
(E. coli) is an opportunistic pathogen in both human and animals, which
is frequently isolated and identified in clinic. UPEC is a common pathogen of
human pyelonephritis and other urinary tract infections. It can result in
persist infections in bladder tissues and cause recurrent urinary tract infections (UTIs). Hemolytic uremic syndrome was
also caused by these pathogens in severe cases (Shah
et al. 2019). With the
fast outbreak of antibiotic-resistant bacteria, especially in UTIs, the
application of bacteriophages has restore attention as an efficient alternative
therapy method for E.coli infection control (LaVergne et al. 2018).
Phage is a type of virus that infects bacteria
and affects the biology and evolution of the host bacteria (Cowley et al.
2015; Fu et al. 2015). Phages
can target and lyse host bacteria. Phages are the most ordinary microbes on earth and
show huge diversity in the host range. Phages are cost-effective, have no
severe negative consequences and are highly toxic to antibiotic-resistant
bacteria compared to conventional drugs (Borysowski
et al. 2011; Wang et al. 2016). Currently, the
problem of bacterial resistance is becoming more and more prominent. Clinical
studies have found that phages have shown good therapeutic effects on a variety
of bacterial infections. Meanwhile, due to their strong specificity, rapid
proliferation and wide source, phages are expected to be used as a new
antibacterial agent to supplement or effectively replace antibiotic therapy of
drug-resistant bacteria (Sarker et al. 2016). The results of
related studies show that phage therapy can reduce the amounts of bacteria in
animals, reduce the mortality rate, and even completely cure the pathogenic
bacterial infections (O'Flaherty et al. 2009; Peng et al. 2018).
Here, a virulent phage isolated from the urine sample of patients who
diagnosed with UTI was identified, and also the biological characteristics of E.
coli bacteriophages were analyzed. This work may lay a solid foundation for
the application of bacteriophages in the therapy of uropathogenic E. coli infections.
Materials and Methods
Sample collection
For this study, both bacteriophages and the bacteria
were collected from the urine samples of patients with UTIs in affiliated
hospital of Jinggangshan university. Strains MC1061, MG1655, DM1187 and strain
Min27 of E. coli K-12 were donated by Professor Yaxian Yan of Shanghai
Jiao Tong University. 22 strains of E. coli U4372 and U4261 (Table 1)
were isolated from urine samples, and staphylococcus aureus ATCC43300
was donated by associate professor Xianqing Deng of Jinggangshan university.
The strains were cultured in THB or LB liquid medium at 37°C.
Isolation and identification of phages
Phages
were isolated from urine samples obtained in hospital and transported under 4ºC.
The urine samples were filtered with the 0.22 μm filter membrane, and the obtained filtrate was successively
added to SM buffer for 10-fold dilution. The supernatants were subjected to
lytic phage evaluation using different E. coli isolates on LB plates.
The commonly used double-layer agar plate method was applied to perform the
plaque formation assay. After overnight (8–12 h) incubation of phage and
indicator bacteria, plaque forming unit on the plate of bacteria culture
indicated the existence of lytic phage. Single plaque of the phage was purified
three times until the edge of the plaque is neat and uniform in size. A single
plaque was selected for amplification and culture. Phage supernatant was
deposited at 4°C for following experiments.
Host range evaluation
Spot
test as well as double agar overlay methods were carried out to evaluate the
host range of phage P4261. In the first place, the phages were isolated using
the indicator bacterium E. coli K-12
MC1061 and the wide host range of P4261 was evaluated with the clinical
isolates. In the spot test, the bacteriophage lysate was spotted onto the
bacterial lawn and its lysis activity was determined by the bacteriophage
removal. This study was performed in triplicates.
Morphological characterization by transmission
electron microscopy (TEM)
To
identify the morphological characters of P4261, the phage sample was stained
negatively by 2% phosphotungstic acid (PTA) and observed under TEM. The phage
particles were stained negative by 2% phosphotungstic acid (PTA) and observed
under transmission electron microscope. Briefly, the purified phage
supernatants were added onto a copper grid and dry in RT for 8 min. 10 μL of 2% PTA was added to the dried
phage particles waiting for 5 min. Lastly, the copper
grid was washed for three times with ultrapure water in order to clean the
redundant stain and observed under TEM (FEI, Hillsboro, USA).
Phage genomic type
analysis
Purified
phage genome was extracted and prepared as described previously (Niu et al.
2014). In order to identify the genome type of the phage genome,
purified phage genome was treated with nuclease of DNase I (20 U/µL), RNase A (5 U/µL), and Mung bean nuclease (20 U/µL) at 37°C for 1 h. Products of digested phage nucleic acid were
analyzed using 1% agarose gel electrophoresis.
Assay of optimal multiplicity of infection (MOI)
Overnight
bacteria cultures of E. coli K-12 strain MC1061 were diluted with LB and
incubated in a shaker at 37°C until reaching an early logarithmic growth phase.
The bacteria were counted as CFU/mL, and mixed with phages according to
different MOIs (0.001, 0.01, 0.1, 1, 10 and 100). After 8 h incubation, the
culture was centrifuged with 6000 rpm for 10 min in a refrigerate centrifuge.
After that, the supernatants were then filtered using 0.22 µm filter. The titer of the phage in the supernatant was directly
evaluated.
One-step growth curve
One-step
growth experiments of the phage were carried out using a modified method
described previously (Pajunen et al. 2000). Briefly,
vB_EcoS_P4261 phage was added to a log phase E. coli K-12 MC1061
bacteria culture at a MOI of 0.1, and allow for incubation for 15 min. The
phage and bacteria mixture were subjected to centrifuge at 12,000 rpm for 60
seconds. The supernatants were discarded, and the sediment of phage and
bacteria cells were suspended with fresh LB and then incubated in a shaker of
180 rpm at 37°C. A small amount of samples were collected at 10 min or 30 min
intervals (0, 10, 20, 30, 40, 50, 60, 90, 120, and 150 min) and the phage titer
of each sample were immediately determined. This assay was repeated at least in
triplicate.
Phage stability
The
stability of phage P4261 was evaluated at different temperatures (40, 50, 60,
70 and 80°C). After incubated in the water of different temperature for 30 min,
the phage aliquots were subject to plaque forming assay to detect the phage
titer. The pH stability of the phage at different pH-values was also detected.
To analyze the chemical stability of the phage, the P4261 was incubated with
different concentration of chloroform (1, 2, and 5%, vol/vol) for 30 min at RT.
Furthermore, P4261 was also exposed to ultraviolet ray for 0, 15, 30, 45, 60,
75, 90, 105 and 120 min, and the sample titer was determined immediately
through a double-layer agar plate method.
Bacteriolytic activity of P4261 in vitro
The
fresh E. coli bacteria culture were diluted 1:100 with LB and incubated
at 37°C until reaching OD600 Value 0.4–0.6. Phage P4261 was added at
a relatively high, medium and low MOI of 100, 1, 0.01 and an identical E.
coli (U3872 and U3519) culture with equivalent volume phage was
tested. The mixture was cultured at 37°C in an incubator shaking
of 160 rpm. The bacteria lytic activity of phages was evaluated by
observing OD600 Value of culture medium at an interval of 30min for
4 h.
Table 1: Lytic
activity of phage vB_EcoS_P4261
|
Strain Type |
Strains |
Lytic Activity |
|
E. coli strain K-12 |
MC1061 |
+++ |
|
MG1655 |
+++ |
|
|
DM1187 |
+++ |
|
|
UPEC isolates |
U3872 |
++ |
|
U3519 |
++ |
|
|
U4469 |
+ |
|
|
U4261 |
- |
|
|
U129 |
- |
|
|
U4196 |
- |
|
|
E. coli strain O157 |
Min27 |
- |
|
P. aeruginosa isolates |
U955 |
- |
|
Isolates of enterobacter
cloacae |
U547 |
- |
|
Staphylococcus aureus |
ATCC43300 |
++ |
Note: +++: Very strong lytic ability; + +: lytic
ability; +: weak lytic ability; -: No lytic ability
Table
2: Determination of
optimal multiplicity of infection (MOI) of phage P4261
|
Number |
Bacteria (CFU/mL) |
Phage (PFU/mL) |
MOI |
Phage Titer (PFU/mL) |
|
1 |
109 |
106 |
0.001 |
1.34 × 108 |
|
2 |
109 |
107 |
0.01 |
4.42 × 1010 |
|
3 |
109 |
108 |
0.1 |
7.26 × 1014 |
|
4 |
109 |
109 |
1 |
1.48 × 1011 |
|
5 |
108 |
109 |
10 |
7.52 × 1010 |
|
6 |
107 |
109 |
100 |
3.21 × 109 |
%201614-1620_files/image002.jpg)
Fig. 1: Morphological characterization and life cycle
of Escherichia phage vB_EcoS_P4261
(A)
Transmission Electron Microscopic (TEM) image of Escherichia phage vB_EcoS_P4261 shows that the phage belongs to Siphoviridae family, (B) Observed plaques of Escherichia phage vB_EcoS_P4261
infecting E. coli K-12 MC1061 using double agar overlay method
Statistical analyses
The
data points were drawn by GraphPad Prism 6.0 software (GraphPad Software, Inc.,
La Jolla, U.S.A.). Data are expressed as mean ± standard deviation (SD).
Results
Phage isolation and determination of host range
Here, a
lytic E. coli phage designated as P4261 from urine sample of UTI patient
in affiliated hospital of Jinggangshan university was isolated. Using E.
coli K-12 strain MC1061 as the indicator strain, the phage plaques of
round, smooth and tidy edge and with 1–2 mm in diameter were observed (Fig.
1A). The phage P4261 had a strong ability to form plaques on E. coli
strains and UTIs isolates, suggesting a potential application of therapeutic
effect in UTIs (Table 1). In addition, the P4261 phage showed relative strong
bacteriolytic activity against a MRSA (methicillin-resistant S. aureus)
strain ATCC43300 (Table 1).
The
phage morphology was observed through TEM analysis and the results demonstrated
that the phage P4261 having the icosahedral head of approximately 40 ± 5.0 nm
as well as long non-contractile tail of approximately 115 ± 5.0 nm in length
(Fig. 1B). Based on the classification of International Committee on Taxonomy
of Viruses (ICTV) (Adams et al. 2014), the phage P4261 was likely to belong to the
family Siphoviridae, Caudovirales considering the morphological
characteristics.
Genome type analysis
The purified phage genome was digested by three kind
of different enzyme of nucleases. The electrophoresis results revealed that
P4261 genome was entirely digested by DNase I, while RNase A or Mung bean
nuclease had no effect on the phage genome (Fig. 2), which indicating that
P4261 was a double-stranded DNA virus.
Determination of MOI
The results showed that when MOI=0.1, the titer of
the phage lysates was the highest (7.26 × 1014 PFU/mL; Table 2),
indicating that the optimal MOI of the phage was 0.1.
One-step growth curve
The phage life cycle is one of the important criteria for evaluating
therapeutic phage lysis activity and determining its infectivity. Accordingly, one-step
growth assay with the presence of indicator E. coli MC1061 was carried
out to evaluate the latency time and burst size of the phage. The phage P4261 was found to have a short latency time of 10 min and the calculated burst size was
117 PFU/ infected cell (Fig. 3) according to Burst Size=phage titer
(PFU/mL) at the end
of burst period/host bacteria concentration at the initial stage of infection
(CFU/mL). It is well known that when phages have a short latency time and a
large amount of phage release from infected host, they are characterized as virulent
phages. The results indicated that E. coli phage P4261 is a virulent
phage with very adaptive therapeutic application.
Phage stability
The
potential therapeutic
application of phage P4261 was determined d by evaluating the phage stabilities
in different conditions. The thermal stability was detected under different
temperatures. It was demonstrated that the activity of phage P4261 was relative
stable under 50°C. Higher temperatures led to a progressive inactivation of
phage. Phage P4261 was totally inactivated when it was heated to a high
temperature of 70°C (Fig. 4A).
The
pH stability was evaluated with different pH (2–13) prepared in SM buffer. The
phage showed a relatively high survival rate under pH value of 6 to 10.
However, under other pH scale, the activity of phage decreased dramatically
(Fig. 4B).
In
addition, the survival rate of phage was affected in the presence of 1, 2 and
5% chloroform. The results showed that chloroform had a significant effect on
the titer of phages. As the concentration of chloroform increased, the survival
rate of phages decreased, suggesting that the phages were sensitive to
chloroform (Fig. 4C).
Ultraviolet
irradiation assay showed that the phage titer dropped sharply after sampling at
an interval of 15 min. With the extension of the irradiation time, the phage
titer decreased, indicating that the phage was sensitive to ultraviolet
radiation, but also had certain resistance (Fig. 4D).
The
study indicated that phage structure might be related to survival under adverse
conditions. Previous studies reported probable correlations between phage
morphology character and its survival rate under adverse circumstances (Wang et al.
2016). It was showed that tailed phages can be stable under harsh
environment (Rabitsch et al. 2004). From the above, the phage stability results
showed a wide range of thermal and pH stability and also strong resistance to
ultraviolet.
Bacteriolytic
activity of P4261
The phage P4261 bacteriolytic
activity was determined in a log-phase culture of Uropathogenic E. coli (UPEC)
strains U3872 and U3519. E. coli K-12
was used as a control. We added the phage lysate into the culture with a
relative high MOI of 100 to avoid the bacteria re-proliferation under low MOI
infection status. Results showed that the growth of these strains was
substantially suppressed directly after phage invasion (Fig. 5). These results
suggested that P4261 was highly effective against UPEC in vitro and
indicated a therapeutic potential in vivo.
Discussion
%201614-1620_files/image004.jpg)
Fig. 2: Agarose gel
electrophoresis image of phage vB_EcoS_P4261 genome digested with nuclease.
Lane M: λ-Hind III digest DNA Marker, Lane 1-3: phage vB_EcoS_P4261 genome digested with RNase
A, DNase I, and Mung Bean
Nuclease, respectively
%201614-1620_files/image006.jpg)
Fig. 3: One-step growth curve of phage
vB_EcoS_P4261 in E. coli. Phage vB_EcoS_P4261 was co-incubated with E.
coli strain cultured at a MOI of 0.1 for 15 min at 37°C. Results are shown
as means ± SD from triplicate experiments. The latent period was short as 10
min which represents interval between the absorption and the beginning of the
initial burst. The burst size was estimated at 117 PFU/ infected cell, which
was the ratio of the final count of liberated phage particles to the initial
count of infected bacterial cells
This study described the isolation, identification and
potential application of bacteriophage against UPEC isolates from UTI patients.
According to the phage morphology determined by TEM analysis, it was named as vB_EcoS_P4261. This phage was homologous to phages
which belong to the family Siphoviridae in morphology based on the
classification rule of ICTV. Therefore, here, vB means viruses Bacteriophage;
Eco: Escherichia coli; S: Siphoviridae. 4261 is the urine sample
number of isolated phages.
%201614-1620_files/image008.jpg)
Fig. 4: Stability evaluations of phage
vB_EcoS_P4261. (A) Thermostability: Phage vB_EcoS_P4261 was incubated at
various temperatures as indicated. an aliquot sample of phage P4261 were
collected after 30 min; (B) pH
stability: Phage vB_EcoS_P4261 was incubated under different pH conditions for
3 h; (C) Chloroform stability: Phage
vB_EcoS_P4261 was treated with chloroform (1, 2, 5%, vol/vol) for 30 min; (D)
Ultraviolet light stability: Phage vB_EcoS_P4261 was exposed to UV light for0,
15, 30, 45, 60, 75, 90, 105 and 120 min. The overall results were expressed as
survival rates, and were titrated immediately using double-layer agar plate
method. Results are shown as means ± SD from triplicate experiments
%201614-1620_files/image010.jpg)
Fig. 5: Bacteriolytic
activity of phage vB_EcoS_P4261 against E. coli K-12 strain and UPEC
isolates in vitro. Early exponential cultures of E. coli K-12
strain MC1061 (A) and UPEC
isolates U3519 (B) and U3872 (C) strains were co-cultured with
vB_EcoS_P4261 phage at different MOIs: Low MOI of 0.01, Medium MOI of 1, and
High MOI of 100, respectively. E. coli cultured with a similar volume of
phage diluent was used as a control. Results are shown as means ± SD from
triplicate experiments
Our study investigated virulent phage vB_EcoS_P4261,
which was found to be capable of infecting E. coli K-12 strains, S.
aureus, and UPEC isolates. Characterization experiments showed that P4261
has many biological features suitable for therapy applications in UTIs
including short latent periods, large burst sizes, wide host range and high
stability. UTIs are considered to be one of the most common bacterial infection
cases in community hospitals (Malik et al. 2020), accounting for
about 40% of nosocomial infections. As a result, the proliferation of
drug-resistant isolates of UPECs has refocused researchers' attention on
antimicrobial approaches to phage therapy. The emergence of broad-spectrum
lactamase (ESBL) and strains which can produce biofilm adds another barrier to
the traditional antibiotic treatment of urinary pathogens, further promoting
the exploration of new treatment measures. Many researchers have reported in
vivo and in vitro bacteriolytic activities of phages, which
indicating a potential therapy application as an antibacterial agent (Capparelli et
al. 2006; Watanabe et al. 2007;
Gu et al. 2012; Yen et al. 2017).
In this study, in order to evaluate the
application potential of phage vB_EcoS_P4261 in vitro, bacteriolytic
activity experiments were performed by apply different MOIs to infect the E.
coli strains. Results demonstrated that the phage substantially inhibit and
lysed the bacteria under a relative medium and high MOI, which indicating a
potential phage therapy candidate for antibiotics-resistance UPEC infections.
Since bacteriophages have strong lysis activity against the pathogen
responsible for diseases, the isolation and identification of phages which can
target pathogenic bacteria is of great importance. The application of
bacteriophages in therapy has been reported as a dramatic method for treating Escherchia
coli, Pseudomonas. aeruginosa, Klebsiella pneumoniae and staphylococcus
aureus infections under different conditions, especially in UTIs (Pallavali et
al. 2017). What’s more, antibiotic resistance has reached dangerous
levels for public health, therefore, alternative methods, for example, phage
therapy, are urgently needed. Our study is part of an ongoing effort by the
science behind phage therapy to promote better understanding behind the use of
phages as therapeutic tools (Manohar et al. 2018).
Conclusion
In the
present study, a phage named as vB_EcoS_P4261 against UPEC, and with efficient
lytic ability was identified. The biological characteristics of P4261 suggest
that it is of great potential for its therapeutic application in bacterial infections,
especially in UTIs.
Acknowledgement
We
acknowledge the financial supports of National Natural Science Foundation of
China (Grant No.31860711), Natural Science Foundation of Jiangxi Province
(Grant No.20192BAB215064), Doctoral Research Project
of Jinggangshan University (Grant No. JZB1819) and Science and Technology
Planning Project of Education Department of Jiangxi Province (Grant No.
GJJ190576).
Author
Contributions
Bin Liu, Xiaolong Qu and Qiang Fu designed the study.
Weiye Wang, Huimin Li, Zijun Tang, Wei Zhang performed experimental work. Li
Guo, Yangming Chen collected the samples. Caihua Dai, Yuqing Wang and Yating Xu
analyzed the data. Bin Liu and Qiang Fu wrote the article.
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