Comparison of Staphylococcus Phage K with Close Phage Relatives Commonly Employed in Phage Therapeutics

The increase in antibiotic resistance in pathogenic bacteria is a public health danger requiring alternative treatment options, and this has led to renewed interest in phage therapy. In this respect, we describe the distinct host ranges of Staphylococcus phage K, and two other K-like phages against 23 isolates, including 21 methicillin-resistant S. aureus (MRSA) representative sequence types representing the Irish National MRSA Reference Laboratory collection. The two K-like phages were isolated from the Fersisi therapeutic phage mix from the Tbilisi Eliava Institute, and were designated B1 (vB_SauM_B1) and JA1 (vB_SauM_JA1). The sequence relatedness of B1 and JA1 to phage K was observed to be 95% and 94% respectively. In terms of host range on the 23 Staphylococcus isolates, B1 and JA1 infected 73.9% and 78.2% respectively, whereas K infected only 43.5%. Eleven open reading frames (ORFs) present in both phages B1 and JA1 but absent in phage K were identified by comparative genomic analysis. These ORFs were also found to be present in the genomes of phages (Team 1, vB_SauM-fRuSau02, Sb_1 and ISP) that are components of several commercial phage mixtures with reported wide host ranges. This is the first comparative study of therapeutic staphylococcal phages within the recently described genus Kayvirus.


Introduction
Staphylococcus aureus (S. aureus) is an opportunistic and important pathogen in clinical and health-care settings causing a wide variety of diseases commonly involving the skin, soft tissue, bone, and joints [1]. It is also a well-known causative agent of prosthetic joint infections (PJI), cardiac device infections, and intravascular catheter infections [1]. S. aureus pathogenicity is due, in part, to its ability to acquire and express a wide array of virulence factors, as well as antimicrobial resistance determinants [2], an example of which involves the acquisition of the staphylococcal cassette chromosome (SCCmec) leading to the development of methicillin resistance in S. aureus [3]. Methicillin-resistant S. aureus (MRSA) was first reported in 1961 [4], and has since been observed to cause serious infections in hospitals worldwide. Reports of MRSA clones resistant to the majority of antibiotics are a growing concern [5]. As such, new treatment options are needed.

Origin of Phages B1 and JA1
Phages B1 and JA1 were isolated from the Fersisi commercial phage mixtures; batch 010112 (B1) and F-062015 (JA1). This product is used in the treatment of staphylococcal and streptococcal infections. For the isolation of B1, phage enrichment was carried out using staphylococcal host cultured from the sonicate fluid of a hospital patient suffering from PJI. DPC5246 was subsequently used as propagating host for B1, as a prophage was encountered in the PJI strain. Phage enrichment in the isolation of JA1 was done using the Cork Institute of Technology (CIT) collection strain S. aureus CIT281189. Both the PJI strain and CIT281189 were insensitive to phage K.

Morphology and Host Range of Phages K, B1 and JA1
Phages B1 and JA1 exhibited typical characteristics of phages belonging to the Myoviridae family, similar to the reported morphology of phage K [26]. All three phages possessed an A1 morphology [28], displaying an icosahedral head as well as a long contractile tail. They also contained a structure previously described as knob-like appendages by O'Flaherty et al. [26], extending from their base plates (likely Antibiotics 2018, 7, 37 3 of 14 "clumped/aggregated" base plate appendices) and clearly visible in Figure 1. Estimations were made on the dimensions of these phages (Table 1). Capsid heights were estimated as 92.9 ± 4.0 nm (B1), 87.0 ± 2.1 nm (JA1) and 92.9 ± 3.8 nm (K). Tail dimension were also estimated as 233.0 ± 4.4 × 23.4 ± 1.2 nm (B1), 231.5 ± 4.7 × 22.7 ± 0.9 nm (JA1), and 227.5 ± 5.5 × 23.8 ± 1.0 nm (K), and base plates/knobs complexes were estimated as 30.1 ± 1.8 × 47.2 ± 3.7 nm (B1), 32.5 ± 7.9 × 45.8 ± 1.4 nm (JA1), and 36.6 ± 5.1 × 41.7 ± 2.6 nm (K). Owing to the similar morphology of all three phages, a host range study was conducted to explore possible differences in host spectra across a number of hospital isolates. Twenty-one of these isolates represented the entire collection of MRSA sequence-types identified in Ireland by the National MRSA Reference Laboratory (Dublin, Ireland), and includes the commonly encountered ST22-MRSA-IV, which has been predominant in Irish hospitals since the late 1990s [29]. The other two S. aureus strains used in this study were included as additional phage propagation strains. Host range was assessed by plaque assay technique on lawns of various MRSA strains listed in Table 2. The efficiency of plaquing (EOP) was used to represent the degree to which each of the phages studied infected all 23 staphylococcal strains. Phage JA1 had the broadest host range, forming plaques on 18 out of the 23 staphylococcal strains examined. B1 also had a broad host range and was capable of forming plaques on 17 isolates (with some in common with the 18 lysed by phage JA1). Phage K had the narrowest host range, forming plaques on only 10 of the isolates (including its propagating strain DPC5246). All 23 staphylococcal strains were effectively lysed by at least one of the three phages, with the exception of E1139 (IV) ST45 and E1185 (IV) ST12, whose EOP were significantly low at 3.88 × 10 −6 and 1.16 × 10 −6 respectively; as well as 3488 (VV) ST8, which was resistant to all three phages. Plaque size ranged from 0.5 mm to 1.5 mm, with a halo occurring in some instances (Table 3 and Supplementary Materials, Figure S1). The wide host range encountered in this study is common among K-like phages and has been reported for other staphylococcal K-like phages, such as JD007, which infected 95% of S. aureus isolates obtained from several hospitals in Shanghai, China [30]. displaying an icosahedral head as well as a long contractile tail. They also contained a structure previously described as knob-like appendages by O'Flaherty et al. [26], extending from their base plates (likely "clumped/aggregated" base plate appendices) and clearly visible in Figure 1. Estimations were made on the dimensions of these phages (Table 1). Capsid heights were estimated as 92.9 ± 4.0 nm (B1), 87.0 ± 2.1 nm (JA1) and 92.9 ± 3.8 nm (K). Tail dimension were also estimated as 233.0 ± 4.4 × 23.4 ± 1.2 nm (B1), 231.5 ± 4.7 × 22.7 ± 0.9 nm (JA1), and 227.5 ± 5.5 × 23.8 ± 1.0 nm (K), and base plates/knobs complexes were estimated as 30.1 ± 1.8 × 47.2 ± 3.7nm (B1), 32.5 ± 7.9 × 45.8 ± 1.4 nm (JA1), and 36.6 ± 5.1 × 41.7 ± 2.6 nm (K). Owing to the similar morphology of all three phages, a host range study was conducted to explore possible differences in host spectra across a number of hospital isolates. Twentyone of these isolates represented the entire collection of MRSA sequence-types identified in Ireland by the National MRSA Reference Laboratory (Dublin, Ireland), and includes the commonly encountered ST22-MRSA-IV, which has been predominant in Irish hospitals since the late 1990s [29]. The other two S. aureus strains used in this study were included as additional phage propagation strains. Host range was assessed by plaque assay technique on lawns of various MRSA strains listed in Table 2. The efficiency of plaquing (EOP) was used to represent the degree to which each of the phages studied infected all 23 staphylococcal strains. Phage JA1 had the broadest host range, forming plaques on 18 out of the 23 staphylococcal strains examined. B1 also had a broad host range and was capable of forming plaques on 17 isolates (with some in common with the 18 lysed by phage JA1). Phage K had the narrowest host range, forming plaques on only 10 of the isolates (including its propagating strain DPC5246). All 23 staphylococcal strains were effectively lysed by at least one of the three phages, with the exception of E1139 (IV) ST45 and E1185 (IV) ST12, whose EOP were significantly low at 3.88 × 10 −6 and 1.16 × 10 −6 respectively; as well as 3488 (VV) ST8, which was resistant to all three phages. Plaque size ranged from 0.5 mm to 1.5 mm, with a halo occurring in some instances (Table 3 and Supplementary Materials, Figure S1). The wide host range encountered in this study is common among K-like phages and has been reported for other staphylococcal K-like phages, such as JD007, which infected 95% of S. aureus isolates obtained from several hospitals in Shanghai, China [30].

Phage Adsorption on Phage Resistant Isolates
While some level of phage insensitivity was encountered against all three phages, phage K was the frequently insensitive virion to the S. aureus strains tested, and thus, was chosen to evaluate whether or not adsorption inhibition played a role in its insensitivity. Phage K was able to adsorb to all phage-insensitive strains to approximately the same extent as the propagating strain DPC5246. This rules out the possibility of adsorption inhibition playing a role in the narrow host range encountered with phage K in comparison to both phages B1 and JA1 (Supplementary Materials, Figure S2). Additionally, adsorption studies with phages B1 and JA1 indicated that adsorption did not play a role in the differences observed.

Genome Comparison between Phages B1, JA1, and K
The genome of phage K is 139,831 bp in size with long terminal repeats (LTRs) of 8486 bp [31]. Genomes of similar sizes were obtained for phages B1 and JA1, these being 140,808 bp and 139,484 bp, respectively. Examination of sequence reads allowed the identification of LTRs for these phages, due to the identification of a region within their genomes with roughly double the average number of reads, these regions being 8076 bp and 7651 bp in size for phages B1 and JA1, respectively. This approach to the determination of terminal repeats has been utilized for a number of phages [32][33][34]. The sequences of all three phages, when analyzed, contained the 12 bp inverted repeat sequences 5 -TAAGTACCTGGG-3 and 5 -CCCAGGTACTTA-3 , which separates the LTRs from the non-redundant part of the phage DNA, and are characteristic of K-like phages [22,35]. Thus, the entire packaged genome sizes are 148,884 bp (B1), 147,135 bp (JA1), and 148,317 bp (K). Phage K possessed 212 ORFs in its genome [31,36], whereas phages B1 and JA1 possessed 219 (Supplementary Materials, Table S1) and 215 ORFs (Supplementary Materials, Table S2) respectively.
Nucleotide pairwise sequence alignment based on BLASTN revealed phages B1 and JA1 (including their LTRs) to be 99% identical to each other, thus can be considered different isolates of the same phage species [37]. On the other hand, phages B1 and JA1 (including their LTRs) showed 95% and 94% identity (respectively) to phage K, placing these phages on the boundary of speciation.
The examination of 100 bp sequences upstream of each ORFs on the non-redundant genome of these phages, using MEME [38], identified 44 and 43 RpoD-like promoters for phages B1 and JA1, respectively. It was observed that these promoters where heavily concentrated in regions with ORFs encoding short hypothetical proteins and those with functions associated with nucleotide metabolism and DNA replication, rather than those associated with virion structure (Supplementary Materials, Tables S3 and S4). A similar finding was also reported with K-like phage vB_SauM-fRuSau02 [21]. Additionally, 30 Rho-independent terminators were identified on the non-redundant genomes for both B1 and JA1 (Supplementary Materials, Tables S5 and S6).
Four ORFs present in phage B1 were observed to be absent in JA1 (Table 4). These ORFs encoded two putative terminal repeat-encoded proteins (PhageB1_009, 016) and two proteins of unknown function (phageB1_202, 203). Although both B1 and JA1 had similar content of ORFs with 1% difference between their genomes, both phages varied in their host range on the S. aureus strains they infected. This variation is likely attributed to the difference encountered in their genome. Additionally, multiple ORFs present in phage K but absent in both B1 and JA1 were encountered ( Figure 2, Table 5). Furthermore, ORFs present in both phages B1 and JA1 but absent in K were also encountered ( Figure 2, Table 6). These ORFs are discussed below.  Figure 2, Table 5). Furthermore, ORFs present in both phages B1 and JA1 but absent in K were also encountered ( Figure 2, Table 6). These ORFs are discussed below.      Seventeen ORFs present in phage K were absent in both phages B1 and JA1, with one additional ORF found not to be shared between JA1 and K. These ORFs are listed in Table 5. No function could be assigned to these with the exception of phageK_190, which based on NCBI conserved domain search possessed a metallophosphatase-like domain (cd07390; E value; 3.94 × 10 −30 ) and is a member of the metallophosphatase (MPP) superfamily. Families within this superfamily of enzymes are functionally diverse, involved in the cleavage of phosphoester bonds, and include Mre11/SbcD-like exonucleases, Dbr1-like RNA lariat debranching enzymes, YfcE-like phosphodiesterases, purple acid phosphatases (PAPs), YbbF-like UDP-2,3-diacylglucosamine hydrolases, and acid sphingomyelinases (ASMases) [39].

Characteristic Features of Phages B1 and JA1 ORFs Absent in Phage K
Eleven ORFs present in both phages B1 and JA1 were absent in phage K (Table 6). No putative function could be assigned to the majority of these ORFs based on BLASTP, InterProScan or HHpred analysis, with the exception of phageJA1_084 (phageB1_087) and phageJA1_152 (phageB1_155), which encoded homing endonucleases interrupting both the terminase large subunit and the DNA repair protein, respectively. These homing endonucleases are site-specific DNA endonucleases capable of initiating DNA breaks leading to repair and recombination event that results in the integration of this endonuclease ORF into a gene that was previously lacking it [40]. The presence of these mobile genetic elements is common among known staphylococcal phages of the subfamily Spounavirinae, and these endonucleases ORFs are known to insert themselves into essential phage genes [21,41]. Additionally, HHpred analysis indicated ORFs PhageJA1_208 and PhageB1_214 to possess remote homology to cell-degrading proteins. The majority of these ORFs were found to be located next to the genome termini of JA1 and B1, with genes located in this region having been previously reported in similar phages to be expressed early in phage development [35]. Such proteins are usually involved in subversion of the host's machinery to aid phage takeover [42,43]. Four additional staphylococcal phages that originate in commercial phage therapeutic mixtures are Team1, vB_SauM-fRuSau02, Sb-1 and ISP, as discussed earlier [19][20][21][22][23][24]. These phages were also reported to possess wide host ranges towards a number of clinically relevant S. aureus strains. Although similar, these phages have several feature differences from each other and from phages B1 and JA1. Comparison of nucleotide identities (BLASTN) with phage K shows that they belong to the genus Kayvirus (Supplementary Materials, Table S7) possessing genomes of similar sizes, apart from Sb-1, being smaller than would be expected, suggesting the genome submission may have been incomplete (Figure 3). Additionally, the arrangement of ORFs is quite similar. Furthermore, tRNA genes of these phages were also examined. All seven phages were found to possess the same four tRNA genes for methionine, tryptophan, phenylalanine, and aspartic acid (Supplementary Materials, Table S8). The eleven ORFs which were present in B1 and JA1 but absent in K (Table 6, Supplementary Materials, Figure S3) were similarly present in Team 1, vB_SauM-fRuSau02, Sb-1 and ISP. And likewise, the ORFs present in K, but absent in both B1 and JA1, were also missing in these phages. However, vB_SauM-fRuSau02 possesses a much shorter putative tail protein (RS_159) of 73 amino acids compared to the phage K counterpart (PhageK_151) of 170 amino acids. Non-hypothetical proteins that differed between these phages were a membrane protein (Phage B1_180, PhageJA1_177, and Phage_170) and an ATPase-like protein (Protein id: CCA65911.1 for phage ISP). Other ORFs that differed among these phages were mostly hypothetical proteins.
genes [21,41]. Additionally, HHpred analysis indicated ORFs PhageJA1_208 and PhageB1_214 to possess remote homology to cell-degrading proteins. The majority of these ORFs were found to be located next to the genome termini of JA1 and B1, with genes located in this region having been previously reported in similar phages to be expressed early in phage development [35]. Such proteins are usually involved in subversion of the host's machinery to aid phage takeover [42,43].
Four additional staphylococcal phages that originate in commercial phage therapeutic mixtures are Team1, vB_SauM-fRuSau02, Sb-1 and ISP, as discussed earlier [19][20][21][22][23][24]. These phages were also reported to possess wide host ranges towards a number of clinically relevant S. aureus strains. Although similar, these phages have several feature differences from each other and from phages B1 and JA1. Comparison of nucleotide identities (BLASTN) with phage K shows that they belong to the genus Kayvirus (Supplementary Materials, Table S7) possessing genomes of similar sizes, apart from Sb-1, being smaller than would be expected, suggesting the genome submission may have been incomplete (Figure 3). Additionally, the arrangement of ORFs is quite similar. Furthermore, tRNA genes of these phages were also examined. All seven phages were found to possess the same four tRNA genes for methionine, tryptophan, phenylalanine, and aspartic acid (Supplementary Materials, Table S8). The eleven ORFs which were present in B1 and JA1 but absent in K (Table 6, Supplementary Materials, Figure S3) were similarly present in Team 1, vB_SauM-fRuSau02, Sb-1 and ISP. And likewise, the ORFs present in K, but absent in both B1 and JA1, were also missing in these phages. However, vB_SauM-fRuSau02 possesses a much shorter putative tail protein (RS_159) of 73 amino acids compared to the phage K counterpart (PhageK_151) of 170 amino acids. Non-hypothetical proteins that differed between these phages were a membrane protein (Phage B1_180, PhageJA1_177, and Phage_170) and an ATPase-like protein (Protein id: CCA65911.1 for phage ISP). Other ORFs that differed among these phages were mostly hypothetical proteins.  [22][23][24], vB_SauM-fRuSau02 [21], Sb-1 [19] and ISP [20]) using currently available annotations employing BLASTN and visualized with Easyfig.
S. aureus employ several defense strategies against viral attack [10,44] and these, such as restriction modification systems [45] and CRISPR-Cas systems [46], may vary from strain to strain. These defenses along with several variations encountered at the genetic level across phages B1, JA1, and K may explain the differences in host ranges observed in this study.

Bacterial Strains, Phage and Growth Requirement
Phages B1 and JA1 were isolated from a commercial phage cocktail purchased from the George Eliava Institute of Bacteriophage, Microbiology and Virology, Tbilisi, Georgia. The MRSA strains utilized in this study were all acquired from the Irish National MRSA Reference Laboratory, Dublin, Ireland [2] with the exception of DPC5246 and CIT281189, which are routine propagation strains utilized in our laboratory [26,36]. These strains were routinely cultured in Brain Heart Infusion broth (BHI; Sigma, St. Louis, MO, USA) at 37 • C with shaking or on BHI plates containing 1.5% (w/v) bacteriological agar (Sigma). All strains were stocked in BHI containing 40% glycerol and stored at −80 • C.

Phage Host Range and Adsorption Study
Host range assay was performed for phages B1, JA1, and K using the plaque assay plating technique (Tables 2 and 3). This was done in triplicate for three independent experiments. The efficiency of plaquing (EOP) was determined by dividing the phage titer on each test strain by the phage titer of the reference strain (S. aureus DPC5246, in the case of phages B1 and K, and S. aureus CIT281189 for phage JA1) [48]. An adsorption assay was performed according to the protocol previously described elsewhere with some modification [49]. Briefly, MRSA strains were grown to an optical density (OD) of 0.2 at 600 nm (estimated cell count at 10 8 colony forming unit (cfu) mL −1 ) and 100 µL of cells were mixed with 100 µL of respective phage titered at approximately 1 × 10 7 PFU/mL for a multiplicity of infection (MOI) of 0.1. The resulting mixtures were incubated at room temperature for 5 min to allow for phage adsorption. The bound phages were separated from the free phages by centrifugation at 14,000 rpm for 5 min. Adsorption of the phage on each strain was determined by subtracting the number of unbound phage (per mL) from the total input PFU/mL. Adsorption efficiency was expressed as a percentage relative to the propagating strain DPC5246.

Transmission Electron Microscopy
Electron microscopic analysis was performed following negative staining of the CsCl gradient prepared phages on freshly prepared carbon films with 2% (w/v) uranyl acetate. Electron micrographs were taken using a Tecnai 10 transmission electron microscope (FEI Thermo Fisher, Eindhoven, the Netherlands) at an acceleration voltage of 80 kV with a MegaView G2 CDD camera (EMSIS, Muenster, Germany).

Phage DNA Isolation
Phage DNA extraction was performed on CsCl purified high titer phages. These were initially treated with MgCl 2 followed by pre-treatment with DNase and RNase for 60 min at 37 • C. Following that subsequent treatment with SDS, EDTA and proteinase K with further incubation for 60 min at 55 • C. DNA extractions were then performed on the pre-treated samples with phenol/chloroform/isoamyl alcohol (25:24:1 v/v/v) and chloroform/isoamyl alcohol (24:1 v/v). DNA precipitation was achieved using sodium acetate and 95% ethanol. DNA quality and quantity were estimated using a Nanodrop (ND-1000) and visualized following agarose gel electrophoresis 3.6. Phage DNA Sequencing DNA sequencing was performed with a high throughput Illumina HiSeq system sequencing (GATC Biotech, Konstanz, Germany). Library preparation was performed by DNA fragmentation together with adapter ligation. The libraries were then measured and quantified on a Fragment Analyzer and then sequenced to generate 2 × 300 bp paired-end reads. De novo assembly was performed using CLC Bio Genomics Workbench v8.0 (Aarhus, Denmark).

Nucleotide Sequence Accession Number
The genome sequence for phages B1 and JA1 were deposited into GenBank under the accession numbers MG656408 and MF405094, respectively.

Conclusions
Host range of three highly similar phages was performed in this study, and it was identified that phages B1 and JA1 from the Fersisi commercial phage mix had a much broader host range in comparison to phage K on a representative Irish bank of clinical MRSA sequence type isolates. Comparisons of their genomes lead to the identification of several ORFs absent in phage K, but present in both phages B1 and JA1. These ORFs were also identified in several other staphylococcal phages sourced from commercial phage mixtures (B1, JA1, Team 1 [22][23][24], vB_SauM-fRuSau02 [21], Sb-1 [19] and ISP [20]), also with a reported wide host range. The exact role of these ORFs is currently unknown. However, these ORFs along with several variations encountered at the genetic level between these phages may, in part, explain their different host range. Unfortunately, information is lacking on the influences of various phage resistance systems, which may be active in Staphylococcus aureus. Phage research also needs to focus more on elucidation of the functions of hypothetical proteins to allow greater understanding of how phages overcome such systems.
Supplementary Materials: The following are available online at http://www.mdpi.com/2079-6382/7/2/37/s1, Figure S1: Plaque morphologies of phages B1, JA1 and K with common morphology types encountered in their host range study to include plaques sizes of 2mm (A), 0.5mm (B) and 1.0mm (C), Figure S2: Staphylococcus phage K adsorption to strains of Staphylococcus aureus resistant to infection by, in comparison host strain DPC5246, Figure S3: Comparison of regions within the genome of phage K to closely related staphylococcal phages, Table S1: Annotation of the staphylococcal phage vB_SauM_B1, Table S2: Annotation of the staphylococcal phage vB_SauM_JA1, Table S3: Predicted Rho-like promoters of Staphylococcus phage B1 genome found using MEME, Table S4: Predicted Rho-like promoters of Staphylococcus phage JA1 genome found using MEME, Table S5: High ∆G rho-independent terminators predicted in the genome Staphylococcus phage B1 identified using ARNold and QuikFold, Table S6: High ∆G rho-independent terminators predicted in the genome Staphylococcus phage JA1 identified using ARNold and QuikFold, Table S7: Percentage similarity based on BLASTN of broad host range Staphylococcus phages that form commercial phage cocktails to that of Staphylococcus phage K, Table S8: tRNA genes of phages B1, JA1, K, vB_SauM-fRuSau02, ISP, Sb-1, Team 1.
Author Contributions: J.A. conducted the majority of lab work and wrote the manuscript, C.B. assisted with bioinformatics, writing, and critically read the manuscript; S.A.M. assisted with the phage host-range work; N.C. provided the Fersisi therapeutic phages and their information, and critically read the manuscript, E.M.G. isolated and helped characterize phage B1, J.OM and O.M. were co-supervisors of J.A. and both critically read the manuscript, H.N. and C.F. were responsible for electron microscopy and phage measurements, and A.C. conceived, funded, guided the study and critically read the manuscript.