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Isolation, characterization, and PCR-based molecular identification of a siphoviridae phage infecting Shigella dysenteriae
Accepted Manuscript Isolation, characterization, and PCR-based molecular identification of a siphoviridae phage infecting Shigella dysenteriae Khashayar Shahin, Hongduo Bao, Majid Komijani, Mohadeseh Barazandeh, Majid Bouzari, Abolghasem Hedayatkhah, Lili Zhang, Hang Zhao, Tao He, Maoda Pang, Ran Wang PII:
S0882-4010(18)31966-1
DOI:
https://doi.org/10.1016/j.micpath.2019.03.037
Reference:
YMPAT 3468
To appear in:
Microbial Pathogenesis
Received Date: 19 November 2018 Revised Date:
26 March 2019
Accepted Date: 27 March 2019
Please cite this article as: Shahin K, Bao H, Komijani M, Barazandeh M, Bouzari M, Hedayatkhah A, Zhang L, Zhao H, He T, Pang M, Wang R, Isolation, characterization, and PCR-based molecular identification of a siphoviridae phage infecting Shigella dysenteriae, Microbial Pathogenesis (2019), doi: https://doi.org/10.1016/j.micpath.2019.03.037. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Isolation, characterization, and PCR-based molecular identification of a Siphoviridae phage infecting Shigella dysenteriae
Khashayar Shahin1, Hongduo Bao1, Majid Komijani2, Mohadeseh Barazandeh1,
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Majid Bouzari3, Abolghasem Hedayatkhah4,5, Lili Zhang1, Hang Zhao1, Tao He1, Maoda Pang1, Ran Wang1,6*
State Key Laboratory Cultivation Base of MOST, Institute of Food Safety and
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1
2
Department of Biology, Faculty of Science, Arak University, Arak 38156-8-8349,
Iran 3
Department of Biology, Faculty of Sciences, University of Isfahan, Hezar Jereeb
Street, 81746-73441, Isfahan, Iran
Department of Marine Microbiology and Biogeochemistry, Royal Netherlands
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Nutrition, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, PR China
Institute for Sea Research and Utrecht University, Den Burg, the Netherlands 5
Department of Freshwater and Marine Ecology, IBED, University of Amsterdam,
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Amsterdam, the Netherlands
Jiangsu University, Zhenjiang, Jiangsu 212013, China
*
Corresponding author
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Ran Wang
State Key Laboratory Cultivation Base of MOST, Institute of Food Safety and Nutrition, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, PR China E-mail address: [email protected]
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Background: Shigella dysenteriae is one of the members of Shigella genus which
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was the main responsible of different Shigellosis outbreaks worldwide. The
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increasing consumption of antibiotics has led to the emergence and spreading of
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antibiotic-resistant strains. Therefore, finding new alternatives for infection control
5
is essential, one of which is using bacteriophages.
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Abstract
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isolated from petroleum refinery wastewater. Phage morphological and genetic
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Materials and Methods: Lytic bacteriophage against Shigella dysenteriae was
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the genome size was estimated, and phage resistance to different temperatures and
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characteristics were studied using TEM, and sequencing, respectively. In addition,
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Results: According to the morphology and genetic results, this phage was named
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vB-SdyS-ISF003. Sequencing of the PCR products revealed that the vB-SdyS-
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ISF003 phage belongs to the species T1virus, subfamily Tunavirinae of family
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Siphoviridae. This was the first detected bacteriophage against S. dysenteriae,
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pH, host range, adsorption rate, and one-step growth were investigated.
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S. dysenteriae. The genome size was about 62 kb. vB-SdyS-ISF003 phage has a
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number of desirable characteristics including the limited host range to S.
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which belongs to the family Siphoviridae. In addition, its host range was limited to
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tolerance −20 to 50 °C, pH tolerance of 7- 9 without significant reduction in the
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dysenteriae, very short connection time, a relatively wide range of temperature
phage titer.
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Conclusion: vB-SdyS-ISF003 is a novel virulent T1virus phage and has the
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appropriate potential for being used in bio controlling of S. dysenteriae in different
23
condition.
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Keywords: Shigella dysenteriae; Bacteriophage; biocontrol; Bacilli dysentery
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Diarrhea cases caused by pathogen bacteria, viruses, and parasites are a public
28
health problem [1]. According to the World Health Organization, at least 80
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million cases of bloody diarrhea and 700 thousand deaths occur every year [2].
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One of the most important causes belong to diarrhea is Shigella spp. of the family
31
Enterobacteriaceae. Shigella is responsible for 5-15 % of diarrhea and 1.1 million
32
deaths worldwide [1]. Developing countries hold for 90% of Shigella infections,
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which most often occurred in children younger than five years old. One of the most
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important species of this genus, which has been introduced as the responsible for a
35
number of Shigellosis outbreaks in Africa, South Asia, and Central America, is
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Shigella dysenteriae [2]. Shigella spp. can be transmitted through direct contact
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with infected person, contaminated food and water. In addition, flies can transmit
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this microorganism. Due to its low infective dose (about 200 bacteria), it can
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spread very fast and therefore, preventing the spread of the bacteria is an important
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task in controlling the infection [2]. Overusing antibiotics resulted in the
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appearance of a wide range of resistant strains.. Hence, finding and developing
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alternative therapeutic methods seem vital [3, 4], in which using lytic
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bacteriophages (phage therapy) is a proper method. Bacteriophages are viruses that
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only infect the bacteria and have specific hosts. The advantages of using
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Background
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predictable changes in the host microflora or environmental microbial community,
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bacteriophages are that these viruses do not change, as well as, impose few and
impose no serious side effects on the infected host body (i.e., human). Recent
48
studies indicated the success of phage therapy in the elimination of different
49
pathogens, e.g. Vibrio cholerae, Staphylococcus aureus, Escherichia coli and
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Klebsiella pneumoniae [5, 6] Moreover, it is shown that Shigella phages have a
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high potential to reduce or eliminate epidemic shigellosis in high-risk regions, and
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antibiotic-resistant bacteria in developed countries [4, 7]. Thus, identifying new
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potential phages that target specific pathogens is a must.
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This study aimed to isolate, identify and investigate morphological, physiological
55
and genomic specifications of new lytic bacteriophage infecting Shigella
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dysenteriae.
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Materials and methods Lytic phage isolation and purification
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Culture Collection 1188; PTCC 1188), the conventional phage isolation protocol
62
was used with slight modifications [5]. Several samples of untreated wastewater
63
and sewage from urban, hospital and livestock (Jiangsu province, China), were
64
collected and used as a source of bacteriophage. The samples were subjected to
65
centrifugation (5000 × g for 10 min), then were filtration through 0.45 µm
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sterilized syringe filter (JinTeng, China). One milliliter of an overnight culture of
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S. dysenteriae was added to 20 ml of 2Χ brain heart infusion (BHI) broth. After 3
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hours incubating (37 °C, shaking at 100 rpm), 20 ml of the filtered wastewater was
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added to the medium and reincubated for 18-24 incubation at the same condition.
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Afterward, the suspension was centrifuged, and then the supernatant was filtered
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(0.45 µm). Presence of specific lytic phage in the suspension was investigated by
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double-agar layer technique [5]. The appearance of clear plaques was considered as
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the existence of S. dysenteriae lytic bacteriophage. In order to execute the
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purification, a single plaque was selected, and phage purification procedure was
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performed in three successive times. The phage was propagated routinely
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according to Sambrook and Russell protocol [8].
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In order to isolate the lytic bacteriophage against S. dysenteriae (Persian Type
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To eliminate the bacterial debris and to obtain a more concentrated phage sample,
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the phage suspension (109 pfu/ml) was centrifuged using Beckman Optima L-80
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XP ultracentrifugation (25000 × g, 120 min), and then the pellet was re-suspended
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in SM buffer. Finally, the phage suspension was placed on a carbon-coated copper
84
grid (Ted Pella Inc., USA) and negative staining was performed using 2%
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phosphotungstic acid (PTA), and the observation was done using a Hitachi
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HT7700 transmission electron microscope (Hitachi, Japan) at an operating voltage
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of 100 kV [4].
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Electron microscopy
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The phage genome was extracted as described previously by Sambrook and
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Russell (2001). Briefly, the high concentration suspension of the phage was
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prepared using ultra-centrifugation in a Beckman Optima L-80 XP ultracentrifuge
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(TYPE 45 Ti rotor, 105 000 g, 3 h, 4 ºC) and was treated with DNase I and RNase-
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A
using
95
phenol/chloroform/isoamyl alcohol method. The extracted genome was treated
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with EcoRI- EcoRV- HindIII and BamHI restriction enzymes based on
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China)
[8].
Then,
the
genome
was
extracted
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Isolation and restriction endonuclease analysis of the phage DNA
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agarose (0.6%) gel electrophoresis of the digested DNA. The size of the resulting
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manufacturer’s standard protocol (Thermo Fisher Scientific, US), followed by
fragments was estimated using SequentiX Gel Analyzer software (Klein Raden,
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Germany) as described previously [9].
101 102
Specific primer design
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With regard to TEM results, the phage’s family was identified. Subsequently,
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several specific primers were designed for further molecular identification of the
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isolated phage, in which all the available whole genome sequences of Shigella
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specific Siphoviridae phages in GenBank were used to identify the protected
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regions of the genome. Eight regions were selected to design specific primers using
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Primer3 software (http://bioinfo.ut.ee/primer3-0.4.0/). The specificity of the
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designed
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was
assessed
using
the
primer-BLAST
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primers
(https://www.ncbi.nlm.nih.gov/tools/primer-blast/). The primers information is
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summarized in Table 1.
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PCR reactions and sequencing
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The designed primers were used for molecular identification of the isolated phage.
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(Accession number: MG049919) and Shigella phage vB_SsoS-ISF002 (Accession
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number: MF093736) were tested as controls to validate the accuracy of the primers
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[4, 7, 10]. Amplification reactions were carried out in a total volume of 25 µl of the
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green master mix (TSINGKE, China) according to the manufacturer's protocol
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The genomes of two Siphoviridae phages, Shigella phage vB_SflS-ISF001
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were subjected to electrophoresis in a 1% agarose gel. Presence and sizes of
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amplicons were analyzed by Gel documentation system (Bio-Rad, USA). All the
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using an Eppendorf thermal cycler (Eppendorf AG, Germany). The PCR products
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before sequencing (ABI DNA analyzer Model 3730xl, TSINGKE, China). The
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PCR products were purified using a QIAquick Gel Extraction Kit (Qiagen, Japan)
sequence results were analyzed using Chromas software (version 2.6.5) and
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Nucleotide
BLAST
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(https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=Blast
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Search&LINK_LOC=blasthome).
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The
sequences
were
deposited
DDBJ/EMBL/GenBank under the accession numbers MH719022 to MH719028.
in
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Host range of the phage was assessed using both standard strains and clinical
133
isolates of a number of gram-positive and gram-negative bacteria (Table 2).
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Standard spot assay was used to study the sensitivity of the bacteria, which
135
presences of a clear plaque was considered as positive [11].
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Determination of the host range
Thermal and pH stability
137 138 139
determined using double-agar layer technique and the changes were measured in
140
comparison to initial titer (108 pfu/ml) as described previously [7]. For pH stability
141
test, the phage sample was added to SM buffer with the different range of pH
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values (from 2 to 13) and incubated for 1 hour at 37 °C. For thermal resistance test,
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the samples were incubated at -20, 4, 25, 40, 50, 60, 70 and 80 °C (pH = 7) for one
144
hour. The control group of pH and thermal stability experiments were pH of 7 and
145
a temperature of 25 °C, respectively [12]. All experiments were done in triplicate.
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In order to determine phage stability, the phage titers after treatments were
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Determination of phage adsorption time
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multiplicity of infection [13] of about 0.1. The suspension was kept at 37 °C.
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A specific volume of phage and bacteria suspension was mixed to obtain a
Samples were taken at one minute intervals, and the changes in the phage titers
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were evaluated using double-agar layer method [14]. This assay was repeated three
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times.
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Burst size and latent period were investigated using analysis of one-step growth
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curve according to Haq et al. Briefly, the phage-bacteria suspension with MOI of
159
about 0.01 was centrifuged at 13000 × g for 1 min, then the pellet was transferred
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to a fresh BHI broth and incubated at 37 °C. Samples were taken in 5 minutes
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intervals (up to 60 min), and the changes in phage titer were determined by the
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double-layer agar method [15]. This step was performed in triplicate.
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One-step growth curve
Statistical analysis
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the results. P values of < 0.05 were considered as statistically significant.
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GraphPad Prism software version 6.1 and One-way ANOVA were used to analyze
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Phage isolation and electron microscopy analysis
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The formation of the plaque was considered as the presence of lytic bacteriophage
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against S. dysenteriae (PTCC 1188). A single plaque with 3 mm in diameter was
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selected, enriched, and used for further experiments. According to Electron
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microscopy results, the isolated phage had an icosahedral head (70 ± 3 nm length
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and 55 ± 3 nm width) which was connected directly to a non-contractile tail (160 ±
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5 nm). Based on the morphological characteristics, this phage was classified as a
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member of Siphoviridae family, Caudovirales order (Figure 1). Following the
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rules of classification and nomenclature of viral isolates and with regard to the
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morphological characteristics of the phage, as the name, vB-SdyS-ISF003 was
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assigned [16].
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Results
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The digestion pattern of the phage genome with EcoRV, EcoRI, HindIII, and
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BamHI showed that the genome was only digested by EcoRV. Moreover, the
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analysis of digestion pattern by SequentiX Gel Analyzer software demonstrated
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that the size of vB-SdyS-ISF003 genome could be around 62000 bp (Figure 2-A).
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Phage restriction endonuclease analysis
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PCR and Sequencing of the products
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Electrophoresis (Figure2-B) and sequencing of PCR fragments confirmed that the
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designed primers were specific for Siphoviridae phages. In addition, the
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sequencing results revealed that the vB-SdyS-ISF003 phage belongs to the species
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T1virus, subfamily Tunavirinae of Siphoviridae family.
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The formation of the plaque was considered as the ability to lyse bacteria by vB-
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SdyS-ISF003. vB-SdyS-ISF003 only lysed S. dysenteriae (PTCC 1188), and no
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infectivity was observed in other tested bacteria, neither gram negative nor positive
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ones. Interestingly, no other Shigella species (S.sonnei, S.flexneri S. boydii) tested
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in this study were lysed by this phage (Table 2).
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Thermal and pH stability
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Bacteriophage host rang
200 201 202
at -20, 4, and 40 °C, these changes were minor (less than 5%). At 50 °C, 17% of
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the phages became deactivated, but this decrease was not significant, but the phage
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titer was significantly decreased at temperatures of 60 with more than 50%
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reduction in activity, and it was dramatically reduced to almost zero at 70 °C and
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complete deactivation at 80 °C (Figure 3-A). In the case of pH stability, the phage
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was fully active at pH 8 and there were no changes in the titer of vB-SdyS-ISF003
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compared to that of pH 7. At the pH of 9, although the activity was measured at
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about 90%, this change was not statistically significant. However, the phage titer
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and activity significantly reduced at other tested pH (5, 6, 10-12), while the activity
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Thermal stability showed that although a decrease in the phage titer was observed
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of 2, 3, and 13 (Figure 3-B).
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was dramatically reduced to almost zero at pH of 4 and ultimately stopped at pHs
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Determination of the phage adsorption time and analysis of one-step growth curve
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Assessment of vB-SdyS-ISF003 adsorption rate indicated that about 87% of the
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phages were connected to the host after 12 minutes, and the highest rate of phage
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connection to the host was about 90%, which occurred at minute 17, which
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indicated the rapid connection of vB-SdyS-ISF003 to the host (Figure 4-A).
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Moreover, analysis of the one-step growth curve indicated that the latent period
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and burst size of the phage were approximately 10 min and 128 ± 12 phages per
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host cell, respectively (Figure 4-B).
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Discussion
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The 1.1 million human deaths worldwide from shigellosis indicates the importance
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Generally, shigellosis is referred to the diarrhea caused by the Shigella species.
of controlling Shigella infection [1].
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causes of diarrhea, especially in children below five years of age. S. dysenteriae, an
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important member of this genus, and a causative agent of shigellosis is responsible
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for several outbreaks over the last decades [2]. Low infectious dose along with its
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high virulence has led to rapid spread of the bacteria, making effective prevention
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and treatment of the infections strategies essential for lowering the intensity of
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outbreaks as well as decreasing the morbidity/mortality rate. Despite the fact that
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most of the bacterial diseases can be treated with antibiotics, many studies
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demonstrated the emergence of antibiotic resistant strains among many pathogens
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Clinical and epidemiologic studies signified that Shigella spp. are among the major
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Salmonella spp., etc as a result of irregular and excessive use of antibiotic agents
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such as Pseudomonas aeruginosa, Klebsiella pneumoniae, E.coli, Shigella spp.,
[4, 17, 18]. Thus, providing an alternative strategy to control or prevent bacterial
240
infections is a must. In this study, we managed to isolate a Shigella specific
241
bacteriophage and evaluated the potential to be used as an alternative to control S.
242
dysenteriae.
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The morphological experiment indicated that the isolated bacteriophage belonged
244
to the family Siphoviridae, making vB-SdyS-ISF003 the first detected Siphoviridae
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the specificity and sensitivity of the primers. The results also demonstrated that the
247
phage should be considered as a T1virus in Tunavirinae subfamily. Therefore,
248
compare to common molecular techniques such as whole or partial genome
249
sequencing, it is crystal clear that using PCR approach with such specific primers
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phage for S. dysenteriae. PCR amplifications and sequencing data demonstrated
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primers are highly recommended as a fast and precise sequence-based
252
classification of similar phages. Bioinformatics investigations showed that the
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range of the genome size in Siphoviridae family is about 17-125 kb, including vB-
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SdyS-ISF003 genome (about 62 kb). Previous studies reported several S.
255
dysenteriae lytic phages from different viral families; e.g., Faruque et al. (2003)
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through much more accessible and time and cost-effective. Thus, these sets of
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Jamal et al., (2014) isolated a phage with a genome size of about 38 kb, belong to
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Myoviridae family [20]. However, the current study reported the first Siphoviridae
259
lytic bacteriophage, which was specific for S. dysenteriae.
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The host range of vB-SdyS-ISF003 was limited to the S. dysenteriae strain used in
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this study and it did not infect either any other phylogenetically close (other
262
Shigella species or Enterobacteriaceae) or far (gram-positive) bacteria used in this
263
study. The high specificity of this phage for a single host is an important remark
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reported a Podoviridae bacteriophage with a genome size of about 41 kb [19]; and
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limited host range means the phage has no effects on normal flora of the host body
266
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for making it a suitable candidate for phage cocktail preparation. In other words,
and make only specific and controlled changes to the microbial community of an
267
environment (especially when environmental control is the case). In comparison
268
with vB-SdyS-ISF003, while Faruque et al. (2003) reported a Podoviridae phage
269
(SF-9) which specifically lysed S. dysenteriae, and did not affect any other tested
270
strains [19]., Jamal et al. (2015) isolated a Myoviridae phage (WZ1) which was
271
capable to infect different strains of S. dysenteriae as well as Achromobacter
272
xylosoxidans and Klebsiella pneumonia [20]. It should be noted that different host
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Wichels et al. (1998) declared that Siphoviridae should only be considered as
275
narrow host ranges phages [21]. In contrast, Shahin et al. (2018) and Hamdi et al.
276
(2017) demonstrated that Siphoviridae phages can be considered as broad host
277
range phages [7, 13]. Therefore, our data is in parallel with Wichels et al. (1998)
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domains are declared for Siphoviridae phages infect Shigella species. For instance,
observations and vB-SdyS-ISF003 should be considered as narrow host range
279
phage. In addition, vB-SdyS-ISF003 seems to be a suitable agent as it can only
280
infect the specific target.
281 282
-20 to 50 °C without any significant reduction in the phage titer; however, the
283
activity was significantly decreased at 60 °C, and it was fully inactivated at
284
temperatures above 70 °C. Ja mal et al. (2015) reported a thermal stability range of
285
37-65 °C and complete inactive at 70 °C for WZ1 phage [20]. Faruque et al. (2003)
286
reported that the majority of SF-9 phage particle were inactivated at temperatures
287
higher than 45 °C [19]. In the case of pH stability, the highest activity of vB-SdyS-
288
ISF003 occurs at pH 7 and 8. The phage was much tolerant toward alkaline than
289
acidic pH [4]. SF-9 phage kept 65-82% of its activity after 6 hours of incubation at
290
pHs of 6-9 [19], while WZ1 phage maintained its lytic function at pHs of 5, 9 and
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11 with a minor alternation compared to pH 7 and was fully deactivated at pHs of
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The stability test demonstrated that the phage was able to tolerate the temperatures
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lysing S.sonnei, was fully deactivated at pH below 4 or above 12, and the
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1 and 3 [20]. Shahin et al.(2018) indicated that vB_SsoS-ISF002 phage, capable of
maximum range of function for this phage was pH 7-9 [4]. These studies
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demonstrated that most of the isolated lytic phages against Shigella spp. tolerate
296
slightly alkaline conditions better than acidic conditions. Moreover, most of these
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phages can tolerate temperatures of less than 50 °C. Overall, vB-SdyS-ISF003 has
298
the potential to be used as an effective biocontrol agent in the environment with pH
299
values ranging from 7-10, and temperatures ranging from −20 to 50 °C. On the
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temperatures, which makes it suitable in biocontrol purposes for ready to eat foods,
302
ice creams, or foods that require a cold chain.
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Another specification of the investigated phage was the slightly short adsorption
304
rate, in which about 90% of the phages were attached to the host after 17 minutes.
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Jamal et al. (2015) reported that the maximum attachment of WZ1 phage to the
306
host cell was 50% after 30 min, and increased to 60% in the presence of 10 mM
307
CaCl2 [20]. Moreover, adsorption rate for vB_SsoS-ISF002 and vB_SflS-ISF001
308
phages had been estimated around 89.7% (10 min) and 87.1% (14 min),
309
respectively [4, 7]. Hence, the high adsorption rate of vB-SdyS-ISF003 is much
310
advantageous than other phages, particularly for potential phage therapy against S.
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dysenteriae.
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other hand, a noteworthy specification of this phage is its tolerance against cold-
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rapid lytic activity and as a result a higher potential for phage therapy purposes.
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One-step growth experiment indicated that vB-SdyS-ISF003 had a relatively large
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Furthermore, large burst size and short latent period of a lytic phage could ensure a
316
period of other Shigella specific phage e.g. vB_SsoS-ISF002 (76 ± 9
317
virions/infected cell, 15 min) and vB_SflS-ISF001 (53 ± 4 virions/ infected cell, 20
318
min) [7 ,4].
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burst size and a short latent period, which was better than burst size and latent
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Conclusion
321
vB-SdyS-ISF003 was the first detected Siphoviridae phage against S. dysenteriae,
322
and showed unique characteristics including limited host range, being activate at a
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relatively wide ranges of temperatures and pH values, short adsorption time, short
324
latent period, and a large burst size. These characteristics ensure the attachment of
325
a large number of phages in a short time on the surface of the host cell, a rapid lytic
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and pH values. Moreover, the high efficiency and accuracy of the PCR-based
328
sequencing may be useful to facilitate further research on bacteriophage. Thus,
329
with regards to the facts mentioned above, this phage is a proper candidate for
330
biocontrolling of S. dysenteriae, and in particular in developing a better and more
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activity and produce a large number of new-borne phages in different temperatures
effective phage cocktail against this pathogen.
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Acknowledgments
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(31702297 and 31701725) and Natural Science Foundation of Jiangsu Province
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This work was supported by National Natural Science Foundation of China
(BK20160577).
References
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[1]Schroeder GN, Hilbi H. Molecular pathogenesis of Shigella spp.: controlling
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host cell signaling, invasion, and death by type III secretion. Clinical Microbiology
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Reviews. 2008;21:134-56.
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[2]Organization WH. Guidelines for the control of shigellosis ,including epidemics due to Shigella dysenteriae type 1. 2005.
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[3]Komijani M, Shahin K, Barazandeh M, Sajadi M. Prevalence of extended-
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spectrum β-lactamases genes in clinical isolates of Pseudomonas aeruginosa.
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Medical Laboratory Journal. 2018;12:34-41.
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[4]Shahin K, Bouzari M, Wang R. Isolation, characterization and genomic
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analysis of a novel lytic bacteriophage vB_SsoS-ISF002 infecting Shigella sonnei
349
and Shigella flexneri. Journal of Medical Microbiology. 2018;67:376-86.
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[5]Komijani M, Bouzari M, Rahimi F. Detection and characterization of a novel
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lytic bacteriophage (vB-KpneM-Isf48) against Klebsiella pneumoniae isolates
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from infected wounds carrying antibiotic-resistance genes (TEM, SHV, and CTX-
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M). Iranian Red Crescent Medical Journal. 2017;19:e34.475
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therapy against bacterial pathogens of aquatic and terrestrial organisms. Viruses.
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2017;9:50.
357
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[6]Doss J, Culbertson K, Hahn D, Camacho J, Barekzi N. A review of phage
[7]Shahin K, Bouzari M. Bacteriophage application for biocontrolling Shigella
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flexneri in contaminated foods. Journal of Food Science Technology. 2018;55:550-
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9.
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[8]Sambrook J, Russell DW. Molecular cloning: a laboratory manual: Cold Spring Harbor Laboratory; 2001.
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[9]Komijani M, Bouzari M, Etemadifar M, Zarkesh-Esfahani H, Shaykh-Baygloo
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N, Ghazimorad A, et al. Torque teno mini virus infection and multiple sclerosis.
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International Journal of Neuroscience. 2011;121:437-41.
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Shigella flexneri vB_SflS-ISF001 bacteriophage. Turkish Journal of Biology.
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2019:43.
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[10]Shahin K, Bouzari M, Wang R. Complete genome sequence analysis of a lytic
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genome sequence of a novel N4-like bacteriophage, pSb-1 infecting Shigella
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boydii. Research in Microbiology. 2014;165:671-8.
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[11]Jun JW, Yun SK, Kim HJ, Chai JY, Park SC. Characterization and complete
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bacteriophage (vB_PmiS-TH) and its application in combination with ampicillin
373
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[12]Yazdi M, Bouzari M, Ghaemi EA. Isolation and characterization of a lytic
against planktonic and biofilm forms of Proteus mirabilis isolated from urinary
374
tract infection. Journal of Molecular Microbiology Biotechnology. 2018;28:37-46.
375
[13]Hamdi S, Rousseau GM, Labrie SJ, Tremblay DM, Kourda RS, Slama KB, et
376
al. Characterization of two polyvalent phages infecting Enterobacteriaceae.
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Scientific Reports. 2017;7:40349.
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[14]Shahrbabak SS, Khodabandehlou Z, Shahverdi AR, Skurnik M, Ackermann
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H-W, Varjosalo M, et al. Isolation, characterization and complete genome
380
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sequence of PhaxI: a phage of Escherichia coli O157: H7. Microbiology.
381
2013;159:1629-38.
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characterization of a virulent bacteriophage IHQ1 specific for Aeromonas punctata
384
from stream water. Microbial Ecology. 2012;63:954-63.
385
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[15]Haq IU, Chaudhry WN, Andleeb S, Qadri I. Isolation and partial
[16]Kropinski AM, Prangishvili D, Lavigne R. Position paper: the creation of a
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rational scheme for the nomenclature of viruses of Bacteria and Archaea.
387
Environmental Microbiology. 2009;11:2775-7.
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antibiotic resistance genes in Escherichia coli isolates from infected wounds.
390
Medical Laboratory Journal. 2017;11:30-5.
391
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[17]Komijani M, Bouzari M, Rahimi F. Detection of TEM, SHV and CTX-M
[18]Scarafile G. Antibiotic resistance: current issues and future strategies. Reviews in Health Care. 2016;7:3-16.
392 393 394
Shigella dysenteriae type 1-specific bacteriophage from environmental waters in
395
Bangladesh. Applied Environmental Microbiology. 2003;69:7028-31.
396
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[19]Faruque SM, Chowdhury N, Khan R, Hasan MR, Nahar J, Islam MJ, et al.
397
new Myoviridae bacteriophage WZ1 against multi‐drug resistant (MDR) Shigella
398
dysenteriae. Journal of Basic Microbiology. 2015;55:420-31.
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[20]Jamal M, Chaudhry WN, Hussain T, Das CR, Andleeb S. Characterization of
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Bacteriophage diversity in the North Sea. Applied Environmental Microbiology.
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[21]Wichels A, Biel SS, Gelderblom HR, Brinkhoff T, Muyzer G, Schütt C.
1998;64:4128.33-
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Table 1
405
Specific primers for detection of Siphoviridae bacteriophage and PCR conditions
406
Amplicon size (bp) 523 643 384
1 cycle: 95 ºC ……. 5 min 35 cycle: 94 ºC ….… 1 min 56 ºC ….… 1 min 72 ºC …..... 1 min
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PCR programs
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Primer Sequence (5́ to 3́) 1 F:CCACGGTAACGAGCATGTTG R:GATGGCTCGGCGTACCGAA 2 F:GACGCAAAAGGCGATCTGTC R:ACCCTTGCCCCCTTGTATCA 3 F:CACCAAAACGCGCAAGAAAATC R:ACGCATCGTCGAGCATAGAA 4 F:GGCTGTATCAGAGGCTACCAAAC R:TCGTCTCCCTCACTACCTCC 5 F:TAACGGCAACTGGCAAAACG R:CGCGATGGCTTCACGGATA 6 F:GGCGATCGCAATTCGTGAAA R:ACCGACCGGGCAAAGAGATA 7 F:AGCCCTGTTAAACCTGGAGC R:AACTCAGGCGCAATGGATCG
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1 cycle: 72 ºC ….... 5 min
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Table 2.
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Bacterial strains which were used in determination of the phage host range
412
Source
Infectivity
PTCC 1188 ATCC 9290
+
Shigella sonnei
Sewage
Shigella sonnei Shigella flexneri
Sewage ATCC 12022
− −
Shigella flexneri
PTCC 1234
Shigella flexneri Escherichia coli
PTCC 1865 ATCC 25922
Escherichia coli
Urinary Tract Infection Bedsore ATCC 13883
Streptococcus pneumoniae
ATCC 49619
Enterobacter cloacae Enterococcus faecalis Salmonella typhimurium Proteus mirabilis
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Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Pseudomonas aeruginosa
−
−
− −
−
− −
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Shigella dysenteriae Shigella sonnei
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Bacterial species
ATCC 27853 Burn Wound Infection Bedsore
ATCC 29212 ATCC 14028
ATCC 43071
ATCC 15305 ATCC 12228
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Staphylococcus saprophyticus Staphylococcus epidermidis
ATCC 35933
Staphylococcus aureus MRSA Bacillus cereus
ATCC 33591 ATCC 11778
Bacillus subtilis
ATCC 12711
Enterobacter aerogene Streptococcus pyogenes
ATCC 13048 ATCC 19615
Proteus mirabilis
ATCC 43071
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Staphylococcus aureus
+,Clear plaque; −, no plaque
−
− −
− − − − − − − − − − − − − −
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Table 3.
415
Analysis of the PCR fragment of the phage
416
MH719022
52%
2
MH719023
42.9%
3
MH719024
45.7%
4
MH719025
48.1%
5
MH719026
47.6%
6
MH719027
48.6%
7
MH719028
47.9%
Putative ORFs hypothetical protein putative tail fiber putative ATPdependent helicase putative tail tape measure protein hypothetical protein putative tail fiber protein putative portal protein
Phage (Accession no.) vB_SsoS-ISF002 (MF093736.1) Sf11 ASKT-2018 (MF468274.1) vB_SsoS-ISF002 (MF093736.1) vB_SsoS-ISF002 (MF093736.1) vB_SsoS-ISF002 (MF093736.1) vB_SsoS-ISF002 (MF093736.1) Sf11 ASKT-2018 (MF468274.1)
Taxonomy Caudovirales; Siphoviridae; Tunavirinae; T1virus Caudovirales; Siphoviridae; Tunavirinae; T1virus Caudovirales; Siphoviridae; Tunavirinae; T1virus Caudovirales; Siphoviridae; Tunavirinae; T1virus Caudovirales; Siphoviridae; Tunavirinae; T1virus Caudovirales; Siphoviridae; Tunavirinae; T1virus Caudovirales; Siphoviridae; Tunavirinae; T1virus
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Best matches in NCBI
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GC content
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Accession no.
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PCR name
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417 418 419 420
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Figure 1.
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Morphology of vB-SdyS-ISF003 phage. A and B, Electron micrographs of vB-
422 423
Figure 2.
424
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SdyS-ISF003 phage.
425
EcoRV , II: EcoRI, III: HindIII, IV: BamHI, V: untreated genomic DNA; B,
426
products of specific PCR to identify Siphoviridae phage. I: vB_SflS-ISF001; II:
427
vB_SsoS-ISF002; III: vB-SdyS-ISF003.
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A, Genomic DNA size of phage vB-SdyS-ISF003. L: Ladder (SinaClon, Iran), I:
Figure 3.
429 430
values. The measures are the mean ± SD (standard deviation) from triplicate assays
431
Figure 4.
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Stability of the phage vB-SdyS-ISF003 at (A) different temperatures and (B) pH
432 433
one step growth curve
434
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Adsorption rate of the vB-SdyS-ISF003 on the surface of S.dysenteriae cells and
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435 436
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vB-SdyS-ISF003 is a novel lytic bacteriophage belongs to Siphoviridae infecting S. dysenteriae. Sequencing of the PCR amplicons demonstrates that vB-SdyS-ISF003 phage belongs to the genus T1virus, subfamily Tunavirinae of family Siphoviridae. Genome length of vB-SdyS-ISF003 is about 62000 bp. Phage vB-SdyS-ISF003 can be introduced as a promising antibacterial agent for S. dysenteriae treatment.
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S0882-4010(18)31966-1
DOI:
https://doi.org/10.1016/j.micpath.2019.03.037
Reference:
YMPAT 3468
To appear in:
Microbial Pathogenesis
Received Date: 19 November 2018 Revised Date:
26 March 2019
Accepted Date: 27 March 2019
Please cite this article as: Shahin K, Bao H, Komijani M, Barazandeh M, Bouzari M, Hedayatkhah A, Zhang L, Zhao H, He T, Pang M, Wang R, Isolation, characterization, and PCR-based molecular identification of a siphoviridae phage infecting Shigella dysenteriae, Microbial Pathogenesis (2019), doi: https://doi.org/10.1016/j.micpath.2019.03.037. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Isolation, characterization, and PCR-based molecular identification of a Siphoviridae phage infecting Shigella dysenteriae
Khashayar Shahin1, Hongduo Bao1, Majid Komijani2, Mohadeseh Barazandeh1,
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Majid Bouzari3, Abolghasem Hedayatkhah4,5, Lili Zhang1, Hang Zhao1, Tao He1, Maoda Pang1, Ran Wang1,6*
State Key Laboratory Cultivation Base of MOST, Institute of Food Safety and
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1
2
Department of Biology, Faculty of Science, Arak University, Arak 38156-8-8349,
Iran 3
Department of Biology, Faculty of Sciences, University of Isfahan, Hezar Jereeb
Street, 81746-73441, Isfahan, Iran
Department of Marine Microbiology and Biogeochemistry, Royal Netherlands
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Nutrition, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, PR China
Institute for Sea Research and Utrecht University, Den Burg, the Netherlands 5
Department of Freshwater and Marine Ecology, IBED, University of Amsterdam,
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Amsterdam, the Netherlands
Jiangsu University, Zhenjiang, Jiangsu 212013, China
*
Corresponding author
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Ran Wang
State Key Laboratory Cultivation Base of MOST, Institute of Food Safety and Nutrition, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, PR China E-mail address: [email protected]
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Background: Shigella dysenteriae is one of the members of Shigella genus which
2
was the main responsible of different Shigellosis outbreaks worldwide. The
3
increasing consumption of antibiotics has led to the emergence and spreading of
4
antibiotic-resistant strains. Therefore, finding new alternatives for infection control
5
is essential, one of which is using bacteriophages.
6
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Abstract
7
isolated from petroleum refinery wastewater. Phage morphological and genetic
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Materials and Methods: Lytic bacteriophage against Shigella dysenteriae was
9
the genome size was estimated, and phage resistance to different temperatures and
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characteristics were studied using TEM, and sequencing, respectively. In addition,
11
Results: According to the morphology and genetic results, this phage was named
12
vB-SdyS-ISF003. Sequencing of the PCR products revealed that the vB-SdyS-
13
ISF003 phage belongs to the species T1virus, subfamily Tunavirinae of family
14
Siphoviridae. This was the first detected bacteriophage against S. dysenteriae,
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pH, host range, adsorption rate, and one-step growth were investigated.
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S. dysenteriae. The genome size was about 62 kb. vB-SdyS-ISF003 phage has a
17
number of desirable characteristics including the limited host range to S.
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which belongs to the family Siphoviridae. In addition, its host range was limited to
19
tolerance −20 to 50 °C, pH tolerance of 7- 9 without significant reduction in the
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dysenteriae, very short connection time, a relatively wide range of temperature
phage titer.
21
Conclusion: vB-SdyS-ISF003 is a novel virulent T1virus phage and has the
22
appropriate potential for being used in bio controlling of S. dysenteriae in different
23
condition.
24
Keywords: Shigella dysenteriae; Bacteriophage; biocontrol; Bacilli dysentery
25 26
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Diarrhea cases caused by pathogen bacteria, viruses, and parasites are a public
28
health problem [1]. According to the World Health Organization, at least 80
29
million cases of bloody diarrhea and 700 thousand deaths occur every year [2].
30
One of the most important causes belong to diarrhea is Shigella spp. of the family
31
Enterobacteriaceae. Shigella is responsible for 5-15 % of diarrhea and 1.1 million
32
deaths worldwide [1]. Developing countries hold for 90% of Shigella infections,
33
which most often occurred in children younger than five years old. One of the most
34
important species of this genus, which has been introduced as the responsible for a
35
number of Shigellosis outbreaks in Africa, South Asia, and Central America, is
36
Shigella dysenteriae [2]. Shigella spp. can be transmitted through direct contact
37
with infected person, contaminated food and water. In addition, flies can transmit
38
this microorganism. Due to its low infective dose (about 200 bacteria), it can
39
spread very fast and therefore, preventing the spread of the bacteria is an important
40
task in controlling the infection [2]. Overusing antibiotics resulted in the
41
appearance of a wide range of resistant strains.. Hence, finding and developing
42
alternative therapeutic methods seem vital [3, 4], in which using lytic
43
bacteriophages (phage therapy) is a proper method. Bacteriophages are viruses that
44
only infect the bacteria and have specific hosts. The advantages of using
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Background
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predictable changes in the host microflora or environmental microbial community,
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bacteriophages are that these viruses do not change, as well as, impose few and
impose no serious side effects on the infected host body (i.e., human). Recent
48
studies indicated the success of phage therapy in the elimination of different
49
pathogens, e.g. Vibrio cholerae, Staphylococcus aureus, Escherichia coli and
50
Klebsiella pneumoniae [5, 6] Moreover, it is shown that Shigella phages have a
51
high potential to reduce or eliminate epidemic shigellosis in high-risk regions, and
52
antibiotic-resistant bacteria in developed countries [4, 7]. Thus, identifying new
53
potential phages that target specific pathogens is a must.
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This study aimed to isolate, identify and investigate morphological, physiological
55
and genomic specifications of new lytic bacteriophage infecting Shigella
56
dysenteriae.
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Materials and methods Lytic phage isolation and purification
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Culture Collection 1188; PTCC 1188), the conventional phage isolation protocol
62
was used with slight modifications [5]. Several samples of untreated wastewater
63
and sewage from urban, hospital and livestock (Jiangsu province, China), were
64
collected and used as a source of bacteriophage. The samples were subjected to
65
centrifugation (5000 × g for 10 min), then were filtration through 0.45 µm
66
sterilized syringe filter (JinTeng, China). One milliliter of an overnight culture of
67
S. dysenteriae was added to 20 ml of 2Χ brain heart infusion (BHI) broth. After 3
68
hours incubating (37 °C, shaking at 100 rpm), 20 ml of the filtered wastewater was
69
added to the medium and reincubated for 18-24 incubation at the same condition.
70
Afterward, the suspension was centrifuged, and then the supernatant was filtered
71
(0.45 µm). Presence of specific lytic phage in the suspension was investigated by
72
double-agar layer technique [5]. The appearance of clear plaques was considered as
73
the existence of S. dysenteriae lytic bacteriophage. In order to execute the
74
purification, a single plaque was selected, and phage purification procedure was
75
performed in three successive times. The phage was propagated routinely
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according to Sambrook and Russell protocol [8].
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In order to isolate the lytic bacteriophage against S. dysenteriae (Persian Type
78 79
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To eliminate the bacterial debris and to obtain a more concentrated phage sample,
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the phage suspension (109 pfu/ml) was centrifuged using Beckman Optima L-80
82
XP ultracentrifugation (25000 × g, 120 min), and then the pellet was re-suspended
83
in SM buffer. Finally, the phage suspension was placed on a carbon-coated copper
84
grid (Ted Pella Inc., USA) and negative staining was performed using 2%
85
phosphotungstic acid (PTA), and the observation was done using a Hitachi
86
HT7700 transmission electron microscope (Hitachi, Japan) at an operating voltage
87
of 100 kV [4].
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Electron microscopy
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The phage genome was extracted as described previously by Sambrook and
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Russell (2001). Briefly, the high concentration suspension of the phage was
92
prepared using ultra-centrifugation in a Beckman Optima L-80 XP ultracentrifuge
93
(TYPE 45 Ti rotor, 105 000 g, 3 h, 4 ºC) and was treated with DNase I and RNase-
94
A
using
95
phenol/chloroform/isoamyl alcohol method. The extracted genome was treated
96
with EcoRI- EcoRV- HindIII and BamHI restriction enzymes based on
97
China)
[8].
Then,
the
genome
was
extracted
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Isolation and restriction endonuclease analysis of the phage DNA
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agarose (0.6%) gel electrophoresis of the digested DNA. The size of the resulting
99
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manufacturer’s standard protocol (Thermo Fisher Scientific, US), followed by
fragments was estimated using SequentiX Gel Analyzer software (Klein Raden,
100
Germany) as described previously [9].
101 102
Specific primer design
103
With regard to TEM results, the phage’s family was identified. Subsequently,
104
several specific primers were designed for further molecular identification of the
105
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isolated phage, in which all the available whole genome sequences of Shigella
106
specific Siphoviridae phages in GenBank were used to identify the protected
107
regions of the genome. Eight regions were selected to design specific primers using
108
Primer3 software (http://bioinfo.ut.ee/primer3-0.4.0/). The specificity of the
109
designed
110
was
assessed
using
the
primer-BLAST
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(https://www.ncbi.nlm.nih.gov/tools/primer-blast/). The primers information is
111
summarized in Table 1.
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PCR reactions and sequencing
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The designed primers were used for molecular identification of the isolated phage.
115 116
(Accession number: MG049919) and Shigella phage vB_SsoS-ISF002 (Accession
117
number: MF093736) were tested as controls to validate the accuracy of the primers
118
[4, 7, 10]. Amplification reactions were carried out in a total volume of 25 µl of the
119
green master mix (TSINGKE, China) according to the manufacturer's protocol
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The genomes of two Siphoviridae phages, Shigella phage vB_SflS-ISF001
121
were subjected to electrophoresis in a 1% agarose gel. Presence and sizes of
122
amplicons were analyzed by Gel documentation system (Bio-Rad, USA). All the
123
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using an Eppendorf thermal cycler (Eppendorf AG, Germany). The PCR products
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before sequencing (ABI DNA analyzer Model 3730xl, TSINGKE, China). The
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PCR products were purified using a QIAquick Gel Extraction Kit (Qiagen, Japan)
sequence results were analyzed using Chromas software (version 2.6.5) and
126
Nucleotide
BLAST
127
(https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=Blast
128
Search&LINK_LOC=blasthome).
129
The
sequences
were
deposited
DDBJ/EMBL/GenBank under the accession numbers MH719022 to MH719028.
in
130 131
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Host range of the phage was assessed using both standard strains and clinical
133
isolates of a number of gram-positive and gram-negative bacteria (Table 2).
134
Standard spot assay was used to study the sensitivity of the bacteria, which
135
presences of a clear plaque was considered as positive [11].
136
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Determination of the host range
Thermal and pH stability
137 138 139
determined using double-agar layer technique and the changes were measured in
140
comparison to initial titer (108 pfu/ml) as described previously [7]. For pH stability
141
test, the phage sample was added to SM buffer with the different range of pH
142
values (from 2 to 13) and incubated for 1 hour at 37 °C. For thermal resistance test,
143
the samples were incubated at -20, 4, 25, 40, 50, 60, 70 and 80 °C (pH = 7) for one
144
hour. The control group of pH and thermal stability experiments were pH of 7 and
145
a temperature of 25 °C, respectively [12]. All experiments were done in triplicate.
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In order to determine phage stability, the phage titers after treatments were
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Determination of phage adsorption time
147 148 149
multiplicity of infection [13] of about 0.1. The suspension was kept at 37 °C.
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A specific volume of phage and bacteria suspension was mixed to obtain a
Samples were taken at one minute intervals, and the changes in the phage titers
151
were evaluated using double-agar layer method [14]. This assay was repeated three
152
times.
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Burst size and latent period were investigated using analysis of one-step growth
158
curve according to Haq et al. Briefly, the phage-bacteria suspension with MOI of
159
about 0.01 was centrifuged at 13000 × g for 1 min, then the pellet was transferred
160
to a fresh BHI broth and incubated at 37 °C. Samples were taken in 5 minutes
161
intervals (up to 60 min), and the changes in phage titer were determined by the
162
double-layer agar method [15]. This step was performed in triplicate.
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One-step growth curve
Statistical analysis
164 165 166
the results. P values of < 0.05 were considered as statistically significant.
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Phage isolation and electron microscopy analysis
170
The formation of the plaque was considered as the presence of lytic bacteriophage
171
against S. dysenteriae (PTCC 1188). A single plaque with 3 mm in diameter was
172
selected, enriched, and used for further experiments. According to Electron
173
microscopy results, the isolated phage had an icosahedral head (70 ± 3 nm length
174
and 55 ± 3 nm width) which was connected directly to a non-contractile tail (160 ±
175
5 nm). Based on the morphological characteristics, this phage was classified as a
176
member of Siphoviridae family, Caudovirales order (Figure 1). Following the
177
rules of classification and nomenclature of viral isolates and with regard to the
178
morphological characteristics of the phage, as the name, vB-SdyS-ISF003 was
179
assigned [16].
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Results
181 182
The digestion pattern of the phage genome with EcoRV, EcoRI, HindIII, and
183
BamHI showed that the genome was only digested by EcoRV. Moreover, the
184
analysis of digestion pattern by SequentiX Gel Analyzer software demonstrated
185
that the size of vB-SdyS-ISF003 genome could be around 62000 bp (Figure 2-A).
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Phage restriction endonuclease analysis
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PCR and Sequencing of the products
188
Electrophoresis (Figure2-B) and sequencing of PCR fragments confirmed that the
189
designed primers were specific for Siphoviridae phages. In addition, the
190
sequencing results revealed that the vB-SdyS-ISF003 phage belongs to the species
191
T1virus, subfamily Tunavirinae of Siphoviridae family.
192 193
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The formation of the plaque was considered as the ability to lyse bacteria by vB-
195
SdyS-ISF003. vB-SdyS-ISF003 only lysed S. dysenteriae (PTCC 1188), and no
196
infectivity was observed in other tested bacteria, neither gram negative nor positive
197
ones. Interestingly, no other Shigella species (S.sonnei, S.flexneri S. boydii) tested
198
in this study were lysed by this phage (Table 2).
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Thermal and pH stability
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Bacteriophage host rang
200 201 202
at -20, 4, and 40 °C, these changes were minor (less than 5%). At 50 °C, 17% of
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the phages became deactivated, but this decrease was not significant, but the phage
204
titer was significantly decreased at temperatures of 60 with more than 50%
205
reduction in activity, and it was dramatically reduced to almost zero at 70 °C and
206
complete deactivation at 80 °C (Figure 3-A). In the case of pH stability, the phage
207
was fully active at pH 8 and there were no changes in the titer of vB-SdyS-ISF003
208
compared to that of pH 7. At the pH of 9, although the activity was measured at
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about 90%, this change was not statistically significant. However, the phage titer
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and activity significantly reduced at other tested pH (5, 6, 10-12), while the activity
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Thermal stability showed that although a decrease in the phage titer was observed
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of 2, 3, and 13 (Figure 3-B).
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was dramatically reduced to almost zero at pH of 4 and ultimately stopped at pHs
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Determination of the phage adsorption time and analysis of one-step growth curve
215
Assessment of vB-SdyS-ISF003 adsorption rate indicated that about 87% of the
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phages were connected to the host after 12 minutes, and the highest rate of phage
218
connection to the host was about 90%, which occurred at minute 17, which
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indicated the rapid connection of vB-SdyS-ISF003 to the host (Figure 4-A).
220
Moreover, analysis of the one-step growth curve indicated that the latent period
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and burst size of the phage were approximately 10 min and 128 ± 12 phages per
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host cell, respectively (Figure 4-B).
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Discussion
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The 1.1 million human deaths worldwide from shigellosis indicates the importance
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Generally, shigellosis is referred to the diarrhea caused by the Shigella species.
of controlling Shigella infection [1].
228 229
causes of diarrhea, especially in children below five years of age. S. dysenteriae, an
230
important member of this genus, and a causative agent of shigellosis is responsible
231
for several outbreaks over the last decades [2]. Low infectious dose along with its
232
high virulence has led to rapid spread of the bacteria, making effective prevention
233
and treatment of the infections strategies essential for lowering the intensity of
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outbreaks as well as decreasing the morbidity/mortality rate. Despite the fact that
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most of the bacterial diseases can be treated with antibiotics, many studies
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demonstrated the emergence of antibiotic resistant strains among many pathogens
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Clinical and epidemiologic studies signified that Shigella spp. are among the major
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Salmonella spp., etc as a result of irregular and excessive use of antibiotic agents
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such as Pseudomonas aeruginosa, Klebsiella pneumoniae, E.coli, Shigella spp.,
[4, 17, 18]. Thus, providing an alternative strategy to control or prevent bacterial
240
infections is a must. In this study, we managed to isolate a Shigella specific
241
bacteriophage and evaluated the potential to be used as an alternative to control S.
242
dysenteriae.
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The morphological experiment indicated that the isolated bacteriophage belonged
244
to the family Siphoviridae, making vB-SdyS-ISF003 the first detected Siphoviridae
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the specificity and sensitivity of the primers. The results also demonstrated that the
247
phage should be considered as a T1virus in Tunavirinae subfamily. Therefore,
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compare to common molecular techniques such as whole or partial genome
249
sequencing, it is crystal clear that using PCR approach with such specific primers
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phage for S. dysenteriae. PCR amplifications and sequencing data demonstrated
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primers are highly recommended as a fast and precise sequence-based
252
classification of similar phages. Bioinformatics investigations showed that the
253
range of the genome size in Siphoviridae family is about 17-125 kb, including vB-
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SdyS-ISF003 genome (about 62 kb). Previous studies reported several S.
255
dysenteriae lytic phages from different viral families; e.g., Faruque et al. (2003)
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through much more accessible and time and cost-effective. Thus, these sets of
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Jamal et al., (2014) isolated a phage with a genome size of about 38 kb, belong to
258
Myoviridae family [20]. However, the current study reported the first Siphoviridae
259
lytic bacteriophage, which was specific for S. dysenteriae.
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The host range of vB-SdyS-ISF003 was limited to the S. dysenteriae strain used in
261
this study and it did not infect either any other phylogenetically close (other
262
Shigella species or Enterobacteriaceae) or far (gram-positive) bacteria used in this
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study. The high specificity of this phage for a single host is an important remark
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reported a Podoviridae bacteriophage with a genome size of about 41 kb [19]; and
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limited host range means the phage has no effects on normal flora of the host body
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for making it a suitable candidate for phage cocktail preparation. In other words,
and make only specific and controlled changes to the microbial community of an
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environment (especially when environmental control is the case). In comparison
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with vB-SdyS-ISF003, while Faruque et al. (2003) reported a Podoviridae phage
269
(SF-9) which specifically lysed S. dysenteriae, and did not affect any other tested
270
strains [19]., Jamal et al. (2015) isolated a Myoviridae phage (WZ1) which was
271
capable to infect different strains of S. dysenteriae as well as Achromobacter
272
xylosoxidans and Klebsiella pneumonia [20]. It should be noted that different host
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Wichels et al. (1998) declared that Siphoviridae should only be considered as
275
narrow host ranges phages [21]. In contrast, Shahin et al. (2018) and Hamdi et al.
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(2017) demonstrated that Siphoviridae phages can be considered as broad host
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range phages [7, 13]. Therefore, our data is in parallel with Wichels et al. (1998)
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domains are declared for Siphoviridae phages infect Shigella species. For instance,
observations and vB-SdyS-ISF003 should be considered as narrow host range
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phage. In addition, vB-SdyS-ISF003 seems to be a suitable agent as it can only
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infect the specific target.
281 282
-20 to 50 °C without any significant reduction in the phage titer; however, the
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activity was significantly decreased at 60 °C, and it was fully inactivated at
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temperatures above 70 °C. Ja mal et al. (2015) reported a thermal stability range of
285
37-65 °C and complete inactive at 70 °C for WZ1 phage [20]. Faruque et al. (2003)
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reported that the majority of SF-9 phage particle were inactivated at temperatures
287
higher than 45 °C [19]. In the case of pH stability, the highest activity of vB-SdyS-
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ISF003 occurs at pH 7 and 8. The phage was much tolerant toward alkaline than
289
acidic pH [4]. SF-9 phage kept 65-82% of its activity after 6 hours of incubation at
290
pHs of 6-9 [19], while WZ1 phage maintained its lytic function at pHs of 5, 9 and
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11 with a minor alternation compared to pH 7 and was fully deactivated at pHs of
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The stability test demonstrated that the phage was able to tolerate the temperatures
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lysing S.sonnei, was fully deactivated at pH below 4 or above 12, and the
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1 and 3 [20]. Shahin et al.(2018) indicated that vB_SsoS-ISF002 phage, capable of
maximum range of function for this phage was pH 7-9 [4]. These studies
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demonstrated that most of the isolated lytic phages against Shigella spp. tolerate
296
slightly alkaline conditions better than acidic conditions. Moreover, most of these
297
phages can tolerate temperatures of less than 50 °C. Overall, vB-SdyS-ISF003 has
298
the potential to be used as an effective biocontrol agent in the environment with pH
299
values ranging from 7-10, and temperatures ranging from −20 to 50 °C. On the
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temperatures, which makes it suitable in biocontrol purposes for ready to eat foods,
302
ice creams, or foods that require a cold chain.
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Another specification of the investigated phage was the slightly short adsorption
304
rate, in which about 90% of the phages were attached to the host after 17 minutes.
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Jamal et al. (2015) reported that the maximum attachment of WZ1 phage to the
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host cell was 50% after 30 min, and increased to 60% in the presence of 10 mM
307
CaCl2 [20]. Moreover, adsorption rate for vB_SsoS-ISF002 and vB_SflS-ISF001
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phages had been estimated around 89.7% (10 min) and 87.1% (14 min),
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respectively [4, 7]. Hence, the high adsorption rate of vB-SdyS-ISF003 is much
310
advantageous than other phages, particularly for potential phage therapy against S.
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dysenteriae.
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other hand, a noteworthy specification of this phage is its tolerance against cold-
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rapid lytic activity and as a result a higher potential for phage therapy purposes.
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One-step growth experiment indicated that vB-SdyS-ISF003 had a relatively large
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Furthermore, large burst size and short latent period of a lytic phage could ensure a
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period of other Shigella specific phage e.g. vB_SsoS-ISF002 (76 ± 9
317
virions/infected cell, 15 min) and vB_SflS-ISF001 (53 ± 4 virions/ infected cell, 20
318
min) [7 ,4].
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burst size and a short latent period, which was better than burst size and latent
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Conclusion
321
vB-SdyS-ISF003 was the first detected Siphoviridae phage against S. dysenteriae,
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and showed unique characteristics including limited host range, being activate at a
323
relatively wide ranges of temperatures and pH values, short adsorption time, short
324
latent period, and a large burst size. These characteristics ensure the attachment of
325
a large number of phages in a short time on the surface of the host cell, a rapid lytic
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and pH values. Moreover, the high efficiency and accuracy of the PCR-based
328
sequencing may be useful to facilitate further research on bacteriophage. Thus,
329
with regards to the facts mentioned above, this phage is a proper candidate for
330
biocontrolling of S. dysenteriae, and in particular in developing a better and more
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activity and produce a large number of new-borne phages in different temperatures
effective phage cocktail against this pathogen.
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Acknowledgments
332 333 334 335
(31702297 and 31701725) and Natural Science Foundation of Jiangsu Province
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This work was supported by National Natural Science Foundation of China
(BK20160577).
References
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[1]Schroeder GN, Hilbi H. Molecular pathogenesis of Shigella spp.: controlling
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host cell signaling, invasion, and death by type III secretion. Clinical Microbiology
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[2]Organization WH. Guidelines for the control of shigellosis ,including epidemics due to Shigella dysenteriae type 1. 2005.
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analysis of a novel lytic bacteriophage vB_SsoS-ISF002 infecting Shigella sonnei
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[5]Komijani M, Bouzari M, Rahimi F. Detection and characterization of a novel
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lytic bacteriophage (vB-KpneM-Isf48) against Klebsiella pneumoniae isolates
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M). Iranian Red Crescent Medical Journal. 2017;19:e34.475
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therapy against bacterial pathogens of aquatic and terrestrial organisms. Viruses.
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2017;9:50.
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[6]Doss J, Culbertson K, Hahn D, Camacho J, Barekzi N. A review of phage
[7]Shahin K, Bouzari M. Bacteriophage application for biocontrolling Shigella
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flexneri in contaminated foods. Journal of Food Science Technology. 2018;55:550-
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9.
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[8]Sambrook J, Russell DW. Molecular cloning: a laboratory manual: Cold Spring Harbor Laboratory; 2001.
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[9]Komijani M, Bouzari M, Etemadifar M, Zarkesh-Esfahani H, Shaykh-Baygloo
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N, Ghazimorad A, et al. Torque teno mini virus infection and multiple sclerosis.
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International Journal of Neuroscience. 2011;121:437-41.
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Shigella flexneri vB_SflS-ISF001 bacteriophage. Turkish Journal of Biology.
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2019:43.
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genome sequence of a novel N4-like bacteriophage, pSb-1 infecting Shigella
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boydii. Research in Microbiology. 2014;165:671-8.
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[11]Jun JW, Yun SK, Kim HJ, Chai JY, Park SC. Characterization and complete
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bacteriophage (vB_PmiS-TH) and its application in combination with ampicillin
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[12]Yazdi M, Bouzari M, Ghaemi EA. Isolation and characterization of a lytic
against planktonic and biofilm forms of Proteus mirabilis isolated from urinary
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tract infection. Journal of Molecular Microbiology Biotechnology. 2018;28:37-46.
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[13]Hamdi S, Rousseau GM, Labrie SJ, Tremblay DM, Kourda RS, Slama KB, et
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al. Characterization of two polyvalent phages infecting Enterobacteriaceae.
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Scientific Reports. 2017;7:40349.
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[14]Shahrbabak SS, Khodabandehlou Z, Shahverdi AR, Skurnik M, Ackermann
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H-W, Varjosalo M, et al. Isolation, characterization and complete genome
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sequence of PhaxI: a phage of Escherichia coli O157: H7. Microbiology.
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2013;159:1629-38.
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characterization of a virulent bacteriophage IHQ1 specific for Aeromonas punctata
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from stream water. Microbial Ecology. 2012;63:954-63.
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[15]Haq IU, Chaudhry WN, Andleeb S, Qadri I. Isolation and partial
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rational scheme for the nomenclature of viruses of Bacteria and Archaea.
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Environmental Microbiology. 2009;11:2775-7.
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antibiotic resistance genes in Escherichia coli isolates from infected wounds.
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[17]Komijani M, Bouzari M, Rahimi F. Detection of TEM, SHV and CTX-M
[18]Scarafile G. Antibiotic resistance: current issues and future strategies. Reviews in Health Care. 2016;7:3-16.
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Shigella dysenteriae type 1-specific bacteriophage from environmental waters in
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Bangladesh. Applied Environmental Microbiology. 2003;69:7028-31.
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[19]Faruque SM, Chowdhury N, Khan R, Hasan MR, Nahar J, Islam MJ, et al.
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new Myoviridae bacteriophage WZ1 against multi‐drug resistant (MDR) Shigella
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dysenteriae. Journal of Basic Microbiology. 2015;55:420-31.
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[20]Jamal M, Chaudhry WN, Hussain T, Das CR, Andleeb S. Characterization of
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Bacteriophage diversity in the North Sea. Applied Environmental Microbiology.
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[21]Wichels A, Biel SS, Gelderblom HR, Brinkhoff T, Muyzer G, Schütt C.
1998;64:4128.33-
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Table 1
405
Specific primers for detection of Siphoviridae bacteriophage and PCR conditions
406
Amplicon size (bp) 523 643 384
1 cycle: 95 ºC ……. 5 min 35 cycle: 94 ºC ….… 1 min 56 ºC ….… 1 min 72 ºC …..... 1 min
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PCR programs
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Primer Sequence (5́ to 3́) 1 F:CCACGGTAACGAGCATGTTG R:GATGGCTCGGCGTACCGAA 2 F:GACGCAAAAGGCGATCTGTC R:ACCCTTGCCCCCTTGTATCA 3 F:CACCAAAACGCGCAAGAAAATC R:ACGCATCGTCGAGCATAGAA 4 F:GGCTGTATCAGAGGCTACCAAAC R:TCGTCTCCCTCACTACCTCC 5 F:TAACGGCAACTGGCAAAACG R:CGCGATGGCTTCACGGATA 6 F:GGCGATCGCAATTCGTGAAA R:ACCGACCGGGCAAAGAGATA 7 F:AGCCCTGTTAAACCTGGAGC R:AACTCAGGCGCAATGGATCG
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1 cycle: 72 ºC ….... 5 min
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Table 2.
411
Bacterial strains which were used in determination of the phage host range
412
Source
Infectivity
PTCC 1188 ATCC 9290
+
Shigella sonnei
Sewage
Shigella sonnei Shigella flexneri
Sewage ATCC 12022
− −
Shigella flexneri
PTCC 1234
Shigella flexneri Escherichia coli
PTCC 1865 ATCC 25922
Escherichia coli
Urinary Tract Infection Bedsore ATCC 13883
Streptococcus pneumoniae
ATCC 49619
Enterobacter cloacae Enterococcus faecalis Salmonella typhimurium Proteus mirabilis
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Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Pseudomonas aeruginosa
−
−
− −
−
− −
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Shigella dysenteriae Shigella sonnei
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Bacterial species
ATCC 27853 Burn Wound Infection Bedsore
ATCC 29212 ATCC 14028
ATCC 43071
ATCC 15305 ATCC 12228
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Staphylococcus saprophyticus Staphylococcus epidermidis
ATCC 35933
Staphylococcus aureus MRSA Bacillus cereus
ATCC 33591 ATCC 11778
Bacillus subtilis
ATCC 12711
Enterobacter aerogene Streptococcus pyogenes
ATCC 13048 ATCC 19615
Proteus mirabilis
ATCC 43071
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Staphylococcus aureus
+,Clear plaque; −, no plaque
−
− −
− − − − − − − − − − − − − −
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413 414
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Table 3.
415
Analysis of the PCR fragment of the phage
416
MH719022
52%
2
MH719023
42.9%
3
MH719024
45.7%
4
MH719025
48.1%
5
MH719026
47.6%
6
MH719027
48.6%
7
MH719028
47.9%
Putative ORFs hypothetical protein putative tail fiber putative ATPdependent helicase putative tail tape measure protein hypothetical protein putative tail fiber protein putative portal protein
Phage (Accession no.) vB_SsoS-ISF002 (MF093736.1) Sf11 ASKT-2018 (MF468274.1) vB_SsoS-ISF002 (MF093736.1) vB_SsoS-ISF002 (MF093736.1) vB_SsoS-ISF002 (MF093736.1) vB_SsoS-ISF002 (MF093736.1) Sf11 ASKT-2018 (MF468274.1)
Taxonomy Caudovirales; Siphoviridae; Tunavirinae; T1virus Caudovirales; Siphoviridae; Tunavirinae; T1virus Caudovirales; Siphoviridae; Tunavirinae; T1virus Caudovirales; Siphoviridae; Tunavirinae; T1virus Caudovirales; Siphoviridae; Tunavirinae; T1virus Caudovirales; Siphoviridae; Tunavirinae; T1virus Caudovirales; Siphoviridae; Tunavirinae; T1virus
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Best matches in NCBI
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GC content
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Accession no.
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PCR name
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Figure 1.
421
Morphology of vB-SdyS-ISF003 phage. A and B, Electron micrographs of vB-
422 423
Figure 2.
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SdyS-ISF003 phage.
425
EcoRV , II: EcoRI, III: HindIII, IV: BamHI, V: untreated genomic DNA; B,
426
products of specific PCR to identify Siphoviridae phage. I: vB_SflS-ISF001; II:
427
vB_SsoS-ISF002; III: vB-SdyS-ISF003.
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A, Genomic DNA size of phage vB-SdyS-ISF003. L: Ladder (SinaClon, Iran), I:
Figure 3.
429 430
values. The measures are the mean ± SD (standard deviation) from triplicate assays
431
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Stability of the phage vB-SdyS-ISF003 at (A) different temperatures and (B) pH
432 433
one step growth curve
434
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Adsorption rate of the vB-SdyS-ISF003 on the surface of S.dysenteriae cells and
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435 436
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vB-SdyS-ISF003 is a novel lytic bacteriophage belongs to Siphoviridae infecting S. dysenteriae. Sequencing of the PCR amplicons demonstrates that vB-SdyS-ISF003 phage belongs to the genus T1virus, subfamily Tunavirinae of family Siphoviridae. Genome length of vB-SdyS-ISF003 is about 62000 bp. Phage vB-SdyS-ISF003 can be introduced as a promising antibacterial agent for S. dysenteriae treatment.
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