Complete genome analysis of the newly isolated Shigella sonnei phage vB_SsoM_Z31

This work describes the characterization and genome annotation of the newly isolated lytic phage vB_SsoM_Z31 (referred to as Z31), isolated from wastewater samples collected in Dalian, China. Transmission electron microscopy revealed that phage Z31 belongs to the family Myoviridae, order Caudovirales. This phage specifically infects Shigella sonnei, Shigella dysenteriae, and Escherichia coli. The genome of the phage Z31 is an 89,355-bp-long dsDNA molecule with a G+C content of 38.87%. It was predicted to contain 133 ORFs and encode 24 tRNAs. No homologs of virulence factor genes or antimicrobial resistance genes were found in this phage. Based on the results of nucleotide sequence alignment and phylogenetic analysis, phage Z31 was assigned to the genus Felixounavirus, subfamily Ounavirinae.


Introduction
Shigella is a genus of Gram-negative, nonmotile bacilli belonging to the family Enterobacteriaceae. It includes four species: Shigella dysenteriae, S. flexneri, S. boydii, and S. sonnei. Shigellosis continues to be a major cause of morbidity and mortality in developing countries and is the most important cause of bloody diarrhea worldwide [1,2]. The World Health Organization (WHO) has reported that Shigella spp. are responsible for an estimated 165 million cases of bacillary dysentery, more than 100 million of which occur in developing countries, causing 1 million deaths annually [3]. The highest rate of Shigella infection (69% of cases) and the highest death rate (61% of deaths) occur in individuals younger than 5 years of age [4]. Among the four species of Shigella, S. sonnei is the most common cause of shigellosis in industrialized regions in Europe, North America, and Australia. Its occurrence is currently expanding in middleincome countries across Asia, Latin America, and the Middle East [5]. Shigella is transmitted by direct contact with an infected person, eating contaminated food, or drinking contaminated water. A wide variety of foods frequently become contaminated with Shigella, including fresh fruits [6], vegetables [7], ready-to-eat foods [8], and meat products [9]. Antibiotics have been used to shorten the duration of shigellosis, but the gradual emergence of multidrug-resistant Shigella spp. has been reported in the last decade [10][11][12]. Thus, there is an urgent need to develop a new strategy to control, inhibit, and eliminate Shigella spp. [13]. Bacteriophages are natural predators of bacteria, and they generally kill a single bacterial strain or subtype of bacteria with high specificity. Bacteriophages have demonstrated potential as antibacterial drugs. In this study, we have sequenced and analyzed the complete genome of a newly isolated S. sonnei phage.

Bacterial strains and growth conditions
The bacterial strains used in this study are listed in Table 1

Phage isolation
Phage vB_SsoM_Z31 (referred to as Z31) was isolated from sewage according to procedures described previously by Zhang et al. [14]. Water samples were collected from the Second Affiliated Hospital of Dalian Medical University in China.

Host range investigation and efficiency-of-plating analysis
Twenty-three bacterial strains, including Shigella, Escherichia coli, and Salmonella (Table 1) were used to determine the lytic capacity of phage Z31 using the spot test method on the basis of its ability to form a lysis zone on lawn cultures of different strains [15]. The purified phage Z31 suspension (10 μl, 10 9 PFU/ml) was spotted directly onto the surface of a bacterial lawn in a culture plate and incubated overnight at 37°C. The plate was then examined for the appearance of clear zones around the phage drop. Efficiency of plating (EOP) was used to evaluate the host spectrum of the phage by testing a variety of bacterial strains (positive spot test). EOP was calculated as the phage titer on the test strains divided by the phage titer on the host bacteria.

Phage DNA purification and sequencing
Phage genomic DNA was extracted from a preparation with a high titer of phage particles (10 10 PFU/ml), using the phenol-chloroform-isoamyl alcohol method as described by Sambrook et al. [17].

Genome analysis
Open reading frames (ORFs) were identified using the Gen-eMark server (http:// topaz. gatech. edu/ GeneM ark/ genem arks. cgi) and the RAST server (http:// rast. nmpdr. org/ rast. cgi dtu. dk/ servi ces/ Virul enceF inder/) were used to identify antimicrobial resistance determinants and potential virulence factors, respectively, in the Z31 genome. The DNA polymerase and large subunit terminase sequences were employed to determine the phylogenetic position and DNA packaging strategies of the phage. The DNA polymerase and large subunit terminase amino acid sequences obtained in this study and those of other phages were selected for multiple alignments using the Clustal W algorithm, and phylogenetic trees were constructed in MEGA7 by the neighbor-joining method. A comparative analysis of the complete genome sequences of Z31 and the other members of the genus Felixounavirus was performed using Easyfig [21].

Results and discussion
A lytic phage against S. sonnei (CGMCC 21535) was isolated from sewage. We named this phage vB_SsoM_Z31 (referred to as Z31). TEM analysis revealed that Z31 has an icosahedral head (60 ± 2 nm) connected to a tail (150 ± 2 nm). Based on these structural features, Z31 was designated as a member of the family Myoviridae, order Caudovirales ( Supplementary Fig. S1) Phage Z31 was found to have a double-stranded DNA genome with a length of 89,355 bp and an overall G+C content of 38.87%. Using the RAST server, we identified 133 ORFs and predicted 100 putative protein coding genes in the genome, 33 of which were functionally assigned. Based on bioinformatic predictions, these ORFs were categorized into four functional modules, including phage structure, host lysis, phage DNA packaging and replication, and hypothetical protein (Supplementary Table S1). Using tRNAscan-SE, Z31 was found to encode 24 predicted tRNAs (Supplementary Table S2), located between positions 73,864 and 79,210. tRNA genes are universally distributed in dsDNA phages, and virulent phages contain more tRNAs than temperate phages, with higher codon usage bias [22]. It is possible that phage-encoded tRNAs enhance translation or compensate for less abundant tRNAs in the host. The large number of tRNAs might enable phages to be translated more efficiently, reduce their latency time, and increase their reproduction rate [23].

Conclusion
We conclude that Z31 is a newly isolated phage that can potentially be used as a therapeutic agent. Nucleotide sequence accession number The GenBank accession number for phage vB_SsoM_Z31 is MN655999.

Declarations
Conflict of interest The authors declare that they have no conflict of interest.
Ethical approval This article does not contain any studies with human participants or animals by any of the authors.