Isolation and Characterization of a Novel Phage SaGU1 that Infects Staphylococcus aureus Clinical Isolates from Patients with Atopic Dermatitis

The bacterium Staphylococcus aureus, which colonizes healthy human skin, may cause diseases, such as atopic dermatitis (AD). Treatment for such AD cases involves antibiotic use; however, alternate treatments are preferred owing to the development of antimicrobial resistance. This study aimed to characterize the novel bacteriophage SaGU1 as a potential agent for phage therapy to treat S. aureus infections. SaGU1 that infects S. aureus strains previously isolated from the skin of patients with AD was screened from sewage samples in Gifu, Japan. Its genome was sequenced and analyzed using bioinformatics tools, and the morphology, lytic activity, stability, and host range of the phage were determined. The SaGU1 genome was 140,909 bp with an average GC content of 30.2%. The viral chromosome contained 225 putative protein-coding genes and four tRNA genes, carrying neither toxic nor antibiotic resistance genes. Electron microscopy analysis revealed that SaGU1 belongs to the Myoviridae family. Stability tests showed that SaGU1 was heat-stable under physiological and acidic conditions. Host range testing revealed that SaGU1 can infect a broad range of S. aureus clinical isolates present on the skin of AD patients, whereas it did not kill strains of Staphylococcus epidermidis, which are symbiotic resident bacteria on human skin. Hence, our data suggest that SaGU1 is a potential candidate for developing a phage therapy to treat AD caused by pathogenic S. aureus. Supplementary Information The online version contains supplementary material available at 10.1007/s00284-021-02395-y.


Introduction
Staphylococcus aureus is a Gram-positive commensal bacterium present in human microbiota, however, it can also act as an opportunistic pathogen causing several infectious diseases, including pneumonia, endocarditis, bacteremia [1][2][3][4], and atopic dermatitis (AD), which is a common inflammatory skin disease that can be caused by abnormal colonization of S. aureus [5,6].
The current treatment for such AD cases includes the use of topical antibiotics; however, the resultant symptomatic improvement is temporary, often resulting in the development of antibiotic resistance [7]. The growth of drugresistant S. aureus, such as methicillin-resistant S. aureus (MRSA), has also been reported on the skin of AD patients [8]. In fact, a previous study in the USA, reported that 80% of patients with AD showed colonization of S. aureus on the skin, 16% of which were identified as MRSA [9].
Another issue associated with antibiotic treatment of AD treatment is the impact on the commensal bacterial community [10,11]. Staphylococcus epidermidis represents the predominant symbiotic bacterium within the human skin microbiota, the presence of which helps recover the skin barrier function by improving the skin microbiota in patients with AD. Moreover, the lipopeptide produced by S. epidermidis enhances the production of antimicrobial peptides on the skin surface of humans and mice; thereby, preventing infection from pathogenic bacteria including S. aureus [12][13][14]. Therefore, an efficient strategy to treat AD that does not affect the human skin microbiota is preferred as an alternative therapy to antibiotic treatment.
Recently, the application of bacteriophages has gained attention as a therapeutic tool for altering the human microbiota [15,16]. In this study, we isolated a phage SaGU1 that specifically targets S. aureus isolated from patients with AD. Here, we describe its genomic information, biophysical stability, and ability to infect previously identified clinical isolates of S. aureus and S. epidermidis from the skin of patients with AD.

Bacterial Strains
All bacterial strains used in this study are listed in Table 1.
Staphylococcus clinical isolates were obtained from Prof. Suzuki's Laboratory, Gifu University, Gifu, Japan. The bacterial species were confirmed based on the analysis of the V3-V4 region in the 16S rRNA gene [17]. All bacterial strains except Listeria innocua KF2492 were grown in lysogeny broth (LB; Formedium, UK) medium at 37 °C. L. innocua was grown in brain heart infusion broth (BHI; BD Difco, NJ).

Drug Susceptibility Testing
Drug susceptibility was determined according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [19] using a DP32 drug plate (Eiken Chemical, Japan).
Briefly, overnight cultures of the selected Staphylococcus isolates were diluted to an OD 600 of approximately 0.25. Next, 25 µL of the diluted bacterial culture was added to 12 mL of LB medium, of which 100 µL was added to each well of the DP32 drug plate. The plate was incubated at 37 °C for 16-20 h. The minimal inhibitory concentrations (MICs) of the strains were determined according to the manufacturer's instructions and CLSI guidelines. MRSA was defined as S. aureus showing an MIC as follows: oxacillin (MPIPC), ≥ 4 μg/mL; cefoxitin (CFX), ≥ 8 μg/mL according to CLSI guidelines.

Isolation and Propagation of Bacteriophages
Phage screening was performed using sewage samples obtained from the northern plant of the Water and Sewage Division of Gifu City, Gifu, Japan, according to the previously published protocol [20]. Briefly, 1.6 L of sewage samples was centrifuged at 8000×g for 20 min at 4 °C, and the resulting supernatant was added to polyethylene glycol 6000 (final concentration, 10% w/v) and NaCl (final concentration, 4% w/v), and stored overnight at 4 °C. The sample was then centrifuged at 10,000×g at 4 °C for 90 min, and the resulting precipitate was re-suspended in 2 mL of phage buffer (10 mM Tris-HCl [pH 7.5], 10 mM CaCl 2 , 10 mM MgSO 4 , 70 mM NaCl). After adding a few drops of chloroform, the sample was kept on ice for 6 h. The sample was then centrifuged at 8000×g for 10 min, filtrated through 0.22 µm filters (Merck Millipore, Ireland), and stored at 4 °C. Next, 100 µL of the solution and 200 µL of an overnight culture of each S. aureus strain were mixed with soft agar, and subsequently overlaid on the LB plate (double-layer plate method). After incubation at 37 °C, the obtained plaques were individually picked up and re-suspended in 100 µL of phage buffer. The titer of the phage lysate was measured by spotting tenfold serially diluted phages on the bacterial lawn and calculated as plaque-forming units (PFU)/mL.

Transmission Electron Microscopy (TEM)
The phage lysate was prepared for TEM according to a previously published method [21]. Samples were negatively stained with 2% phosphotungstic acid (w/v, pH 7.0) and observed using a JEM-1200EXII electron microscope (JEOL, Japan). Micrographs were taken at an accelerating voltage of 80 kV.

One-Step Growth Curve
An one-step growth assay was performed according to a previously published method [22]. Bacterial culture (10 6 colony-forming units (CFU)/mL) of S. aureus 1056-1 was mixed with phage lysate at a multiplicity of infection (MOI) of 0.001, and then incubated for 10 min at 37 °C. The mixture was centrifuged at 7000×g for 10 min at 4 °C. The supernatant was discarded, and the pellet was washed twice with LB and subsequently re-suspended in an equal volume of LB. Next, the resuspension was incubated at 37 °C with constant agitation at 250 rpm. Samples were collected every 10 min to measure the phage titers. The burst size was calculated by dividing the average titers for the post-burst time points by the average initial titers.

Thermal and pH Stability Analysis
The

Phage Host Range
The host range of SaGU1 was determined using the bacterial strains listed in Table 1. Two hundred microliters of stationary phase culture of the host bacteria was mixed with 0.6% soft agar and layered on the LB plate. Next, 2.5 µL of tenfold serially diluted SaGU1 lysate was spotted on a plate in which the host bacteria were overlaid and incubated overnight at 37 °C. The efficiency of plating (EOP) was calculated by dividing the PFU for target bacteria by the PFU for host bacteria.

Genome Sequencing and Bioinformatics Analysis
The phage genome was extracted as described previously [23]. Genome sequencing was performed using the Illumina MiSeq platform (Illumina, USA) and the MiSeq Reagent Kit v3 (Illumina). DNA libraries were prepared using the Nextera XT DNA Preparation Kit (Illumina) for paired-end analyses. Obtained reads were quality-filtered and assembled into contigs and scaffolds using SPAdes 3.9.0 (St. Petersburg State University, Russia) [24]. Prediction of the genes present in phage genomes was carried out using Glimmer [25] in the RAST annotation pipeline [26]. Automatic annotations were manually curated using BLASTp searches against the NCBI non-redundant protein database and NCBI Refseq viral database, with the cutoff level set to an e value < 10 −4 . Prediction of transmembrane helices was conducted using the TMHMM Server ver. 2.0 [27,28]. The terminal repeats region of SaGU1 were identified by Bowtie2 then visualized in Geneious Prime 2020.2.5 (https ://www.genei ous. com) [29].
The phylogenetic tree was generated based on the 17 Staphylococcus phage genomes with PhyML [30]. Bootstrap confidence values (100 resamplings) are as indicated on the internal branches [31].

Nucleotide Sequence Accession Number
The complete SaGU1 genome data has been deposited in the NCBI database under accession number LC574321.

Statistical Analysis
TEM analysis, one-step growth assay, and the stability analysis data were presented as the mean ± standard deviation (SD) and analyzed using GraphPad Prism version 8.4.3 (471) (GraphPad Software, USA).

Isolation of Phage SaGU1 Infecting S. aureus Clinical Isolates
We obtained sewage samples from the northern plant of the Water and Sewage Division of Gifu City, Gifu, Japan, to screen for phages. Two clinical strains of S. aureus (1056-1 and 158-F1) previously isolated from patients with AD were used as host bacteria to screen for the phages. In the screening process, we isolated five and three independent phages from lawns of the strains 1056-1 and 158-F1, respectively. We extracted the genomic DNA from these eight phages to determine their whole genome sequences. All of these isolates showed 100% identical genome sequences; therefore, we selected a single phage infecting 1056-1 as the representative and named it SaGU1.

Characterization of the SaGU1 Genome
SaGU1 is a Class III Staphylococcus phage with a 140,909 bp genome and an overall GC content of 30.2% (Fig. 1a) [32]. The terminal repeats region of SaGU1 was identified in the 98,212-108,279 nucleotide region (Fig. 1b) [33]. The SaGU1 genome encodes a total of 225 predicted genes and four transfer RNAs (tRNAs). The coding density of the genome was 91%, leaving a very small intergenic region. All predicted gene products were searched against the Refseq protein database; however, only 70 coding sequences (CDSs) had previously assigned functions (Supplementary Table S1).
Topologically, the genome of SaGU1 can be divided into two unequal regions; majority of predicted genes were located on the forward strand, whereas all tRNA genes were on the reverse strand. The tRNA genes were separated into two locations: Met-tRNA-CAT and pseudo-tRNA-CCA, Phe-tRNA-GAA, and Asp-tRNA-GTC (Fig. 1a). This arrangement was conserved in other similar Staphylococcus phages. The presence of tRNA in phage genomes is not uncommon, since it has been hypothesized that viral tRNA compensates for the difference in codon usage bias between a phage and its bacterial host, and that the tRNAs correspond to codons that may be inefficiently translated by the host translational machinery [34].
Phylogenetic analysis was then performed based on the whole genome sequences of SaGU1 and other Staphylococcus phages from Cluster C (Fig. 2) [45]. SaGU1 was observed to cluster specifically with the phages of subcluster C1, which generally have a broad host range and therapeutic potential [46].  The gene products with predicted functions encoded modules for virion structure, nucleotide replication and metabolism, and lysis. The structural modules were primarily located between bases 822 and 44,232. However, two genes that encode major tail proteins (SaGU1_71 and SaGU1_72) were located approximately 30 kb upstream from other structural genes, similar to members of kayviruses, such as GH15, MCE-2014, and phILA-RODI [40,41,47]. Moreover, the SaGU1 genome possessed genes encoding structural proteins typically present in members of Myoviridae, including tail sheath protein (SaGU1_20), tail tube protein (SaGU1_21), and baseplate protein (SaGU1_36) which controls its contractile tails [48].
The lysis module of SaGU1 consisted of two adjacent genes, lysin (SaGU1_216) and holin (SaGU1_217). Based on the in silico prediction, the latter was a member of class II holins, which contain two transmembrane helical domains, with both the N-and C-termini present in the cytoplasm [49]. This module was located downstream of the possible pseudo-tRNA, Phe-tRNA, and Asp-tRNA sequences. As there were no lysogeny-related genes detected in the genome, phage SaGU1 likely depends on the lytic cycle to replicate.

Morphology of SaGU1
TEM analysis confirmed that SaGU1 possessed an icosahedral head with a diameter of 86.7 ± 5.0 nm (n = 3), and a contractile tail with a length of 222.7 ± 1.9 nm (n = 3) and a width of 19.3 ± 0.7 nm (n = 3) (Fig. 3a). The myovirus tail is concentric with a tail tube inside a tail sheath. The contracted tail sheath is 95.0 ± 5.0 nm (n = 3) in length, which is approximately 42.7% of the length before contraction, and a 72.5 ± 2.5 nm (n = 3) tail tube protrudes from under the  (Fig. 3b). SaGU1 contains a double base plate (Fig. 3b) and no tail fibers, but globular structures at the tail tip (Fig. 3a). A particle appearing with a black head is empty with no DNA (Fig. 3b). When a myovirus infects a host bacterium, the tail contracts and the DNA in the head is ejected. Moreover, the shape of the whole virus resembled that of other staphylococcal phages, Team1 (NC_025417) [50] and Remus [35]. These morphological characteristics indicate that the SaGU1 phage belongs to the genus Twortlike phages of the family Myoviridae [51,52].

Life Cycle of SaGU1
An one-step growth experiment was performed to analyze the life cycle of SaGU1 (Fig. 4a). The latent phase was found to be 40 min, followed by a 50 min growth phase. A growth plateau was reached within 90 min. The burst size of SaGU1 was calculated as 117 ± 24 PFU/cell. The life cycle of SaGU1 was comparable to those of other Staphylococcus phages in the Myoviridae family, Stau2 (NC_030933) (100 PFU/cell), and IME-SA1 (NC_047729) (80 PFU/cell) [53,54].

Biophysical Stability of SaGU1
Considering that stable phages are required for phage therapy [55], we examined whether SaGU1 is stable at various temperatures and pH values. Thermostability tests showed that SaGU1 was stable between 4 and 40 °C, with a gradual decrease observed between 50 and 70 °C, and complete inactivation achieved at 80 °C (Fig. 4b). The effects of high and low pH on the stability of SaGU1 were also examined (Fig. 4c). SaGU1 showed stable lytic activity following incubation at conditions between pH 4 and pH 10, whereas it lost significant lytic activity after incubation at conditions below pH 2 and above pH 12. The human skin has a pH of approximately 4.1 to 5.8, with a slightly higher pH in the AD patients (approximately pH 5.5); whereas, a pH of 7.5 is optimal for S. aureus growth [56,57]. These results show that SaGU1 application will be stable on the skin of patients with AD, making it potentially useful for phage therapy.

Host Specificity of SaGU1
The host specificity of SaGU1 was examined using Staphylococcus strains listed in We additionally obtained a total of seven known clinical isolates of S. epidermidis (two strains from healthy people Fig. 3 The electron micrographs of phage SaGU1. Pictures were taken by TEM. a SaGU1 normal particle. b SaGU1 particle with the tail contracted. A characteristic feature of the Myoviridae family involves the contraction of the tail sheath, and protrusion of the tail tube from the tip of the tail. A particle with a black head is an empty particle with no DNA. Scale bar, 200 nm and five strains from patients with AD), belonging to different genomic lineages based on the MLST analysis, to assess whether SaGU1 infects S. epidermidis. The results of EOP assays showed that SaGU1 did not infect any of the S. epidermidis strains (EOP < 10 −8 ). In addition, SaGU1 did not infect any other Gram-positive or Gram-negative bacteria ( Table 1).
Taken together, these data indicate that SaGU1 is a staphylococcal phage that specifically infects S. aureus strains of different STs but not S. epidermidis. It may, therefore, by useful to establish the host determinants of SaGU1 in future studies.

Conclusion
A novel Staphylococcus phage SaGU1, which was stable under specific physiological and acidic conditions, was identified and its complete genomic sequence was determined in this study. Given that SaGU1 can specifically infect S. aureus from patients with AD, but not S. epidermidis, it may be a strong candidate for developing phage therapy to treat AD.

Data Availability
The complete genome data of SaGU1 has been deposited in the NCBI database under accession number LC574321.

Compliance with Ethical Standards
Conflict of interest The authors declare that they have no conflict of interest.

Informed Consent
The authors grant the publisher consent to publish the study.
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