Bacteriophage Cocktail Comprising Fifi044 and Fifi318 for Biocontrol of Erwinia amylovora.

Erwinia amylovora is a plant pathogen that causes fire blight on apples and pears. Bacteriophages, which are viruses that selectively infect specific species of bacteria and are harmless to animal cells, have been considered as biological control agents for the prevention of bacterial pathogens. In this study, we aimed to use bacteriophages that infect E. amylovora as biocontrol agents against fire blight. We isolated bacteriophages Fifi044 and Fifi318 infecting E. amylovora, and characterized their morphology, plaque form, and genetic diversity to use as cocktails for disease control. The stabilities of the two phages were investigated at various temperatures and pH values and under sunlight, and long-term storage experiment was conducted for a year. To evaluate whether the two phages were suitable for use in cocktail form, growth curves of E. amylovora were prepared after treating the bacterial cells with single phages and a phage cocktail. In addition, a disease control test was conducted using immature apples and in vitro cultured apple plantlets to determine the biocontrol effects of the phage cocktail. The two phages were morphologically and genetically different, and highly stable up to 50°C and pH value from 4 to 10. The phages showed synergistic effect when used as a cocktail in the inhibition of host bacterial growth and the disease control. This study demonstrated that the potential of the phage cocktail as a biocontrol agent for commercial use.

Novel Single Nucleotide Variations Alter Pathogenicity in Korean Isolates of Erwinia amylovora.

Erwinia amylovora, the causal agent of fire blight disease, has become a serious threat to the pome fruit industry in Korea since 2015. In this study, we showed that two new isolates of E. amylovora, Ea17-2187 and Ea19-7, obtained from pear orchards in Anseong, Korea, exhibited unique pathogenicity compared with other isolates thus far. Both were nonpathogenic to immature apple fruits but occasionally caused disease on immature pear fruits at varying reduced rates. Bioinformatic analyses revealed that their genomes are highly similar to those of the type strains TS3128 and ATCC49946 but have different mutations in essential virulence regulatory genes. Ea17-2187 has a single nucleotide substitution in rcsC, which encodes the core components of the Rcs system that activates the exopolysaccharide amylovoran production. In contrast, Ea19-7 contains a single nucleotide insertion in hrpL, which encodes a master regulator of the type III secretion system. In both cases, the mutation can cause premature termination and production of truncated gene products, disrupting virulence regulation. Introduction of the nonmutated rcsC and hrpL genes into Ea17-2187 and Ea19-7, respectively, fully recovered pathogenicity, comparable with that of TS3128; hence, these mutations were responsible for the altered pathogenicity observed. Interestingly, virulence assays on immature pear fruits showed that the hrpL mutant of Ea19-7 was still pathogenic, although its virulence level was markedly reduced. Taken together, these results suggest that the two new isolates might act as opportunistic pathogens or cheaters and that some Korean isolates might have evolved to acquire alternative pathways for activating pathogenicity factors.

Optimizing the formulation of Erwinia bacteriophages for improved UV stability and adsorption on apple leaves.

Fire blight is a bacterial disease that affects plants of the Rosaceae family and causes significant economic losses worldwide. Although antibiotics have been used to control the disease, concerns about their environmental impact and the potential to promote antibiotic resistance have arisen. Bacteriophages are being investigated as an alternative to antibiotics; however, their efficacy can be affected by environmental stresses, such as UV radiation. In this study, we optimized the formulation of Erwinia phages to enhance their stability in the field, focusing on improving their UV stability and adsorption using adjuvants. Our results confirmed that 4.5 % polysorbate 80 and kaolin improve phage stability under UV stress, resulting in an 80 % increase in PFU value and improved UV protection efficacy. Adsorption assays also demonstrated that polysorbate 80 and kaolin improved the absorption efficiency, with phages detected in plant for up to two weeks. These findings demonstrate the effectiveness of the auxiliary formulation of Erwinia bacteriophages against environmental stress.

Biological and Genetic Characterizations of a Novel Lytic ΦFifi106 against Indigenous Erwinia amylovora and Evaluation of the Control of Fire Blight in Apple Plants.

Erwinia amylovora is a devastating phytobacterium causing fire blight in the Rosaceae family. In this study, ΦFifi106, isolated from pear orchard soil, was further purified and characterized, and its efficacy for the control of fire blight in apple plants was evaluated. Its genomic analysis revealed that it consisted of 84,405 bp and forty-six functional ORFs, without any genes encoding antibiotic resistance, virulence, and lysogenicity. The phage was classified into the genus Kolesnikvirus of the subfamily Ounavirinae. ΦFifi106 specifically infected indigenous E. amylovora and E. pyrifoliae. The lytic activity of ΦFifi106 was stable under temperature and pH ranges of 4-50 °C and 4-10, as well as the exposure to ultraviolet irradiation for 6 h. ΦFifi106 had a latent period of 20 min and a burst size of 310 ± 30 PFU/infected cell. ΦFifi106 efficiently inhibited E. amylovora YKB 14808 at a multiplicity of infection (MOI) of 0.1 for 16 h. Finally, the pretreatment of ΦFifi106 at an MOI of 1000 efficiently reduced disease incidence to 37.0% and disease severity to 0.4 in M9 apple plants. This study addressed the use of ΦFifi106 as a novel, safe, efficient, and effective alternative to control fire blight in apple plants.

First Report of Fire Blight on Chinese hawthorn (Crataegus pinnatifida Bunge) Caused by Erwinia amylovora in Korea.

Fire blight is one of the destructive plant diseases caused by Erwinia amylovora and causes enormous economic losses worldwide. Fire blight was initially reported in apples, pears, and Chinese quince (Park et al. 2016; Myung et al. 2016a, 2016b) in Korea, but recent studies have reported new hosts such as apricot (Lee et al. 2021) and mountain ash (Lim et al, 2023). These reports indicate that fire blight is likely to disperse to new hosts in Korea. During the nationwide survey in June 2021, we observed typical symptoms of blossom blight and shoot blight on a Chinese hawthorn (Crataegus pinnatifida Bunge) just near an orchard (37°09'21.7"N, 127°35'02.6"E) in Icheon, Gyeonggi Province, where fire blight of Asian pear occurred. For identifying its causal agent, bacterial isolates were recovered after incubating at 28 ℃ for 24 hours on tryptic soy agar (TSA) medium (BD Difco, USA) from blighted leaves and shoots that were surface sterilized with 70% alcohol for 30 sec and homogenized in 500 µl of 10mM MgCl2. Pure cultures of white to mucoid colonies were grown on mannitol glutamate yeast extract (MGY) medium, a semi-selective medium for E. amylovora (Shrestha et al, 2003). Two isolates produced 1.5 kb amplicon through colony PCR using amsB primers (Bereswill et al. 1995). Two strains (CPFB26 and CPFB27) from the Chinese hawthorn produced amplicons identical to that from the TS3128 strain of E. amylovora, isolated from the pear tree and identified in 2016 (Park et al. 2016). For the partial 16s rRNA sequences, the total DNA of these two strains was extracted using the Wizard DNA prep kit (Promega, USA), and PCR was performed using fD1 (5'-AGAGTTTGATCCTGGCTCAG-3') and Rp2 (5'-ACGGCTACCTTGTTACGACTT-3') primer sets and further sequenced (Weisburg et al. 1991). These sequences belonged to the E. amylovora clade and were identified as E. amylovora in phylogenetic analysis (GenBank accession no. OP753569 and OP753570). Based on BLASTN analysis, CPFB26 and CPFB27 showed 99.78% similarity to the sequences of the E. amylovora strains TS3128, CFBP 1430, and ATCC 49946. To confirm pathogenicity of the isolates, 10 ㎕ bacterial suspensions (1.5 ⅹ 108 CFU/ml) was injected through the veins of the upper 2nd leaf of 3-month-old clone of apple rootstock (Malus domestica cv. M29) and incubated for six days at 28 ℃ in a chamber with 12 hours of light per day. Petioles and stems turned red hue, and the shoots finally blighted. To complete Koch's postulates, colonies were recovered on TSA medium from the inoculated apple rootstocks and verified through colony PCR for the amsB and A/B primer set (Powney et al. 2011). Hawthorn has been reported as an epidemiologically important alternate host plant of fire blight (van der Zwet et al. 2012). This study is the first to report fire blight caused by E. amylovora in Chinese hawthorn in Korea. Because Chinese hawthorn is natively distributed in Korea and is widely used as a landscaping tree (Jang et al. 2006), the findings of this study suggest that early monitoring could prevent the spread of fire blight through natural hosts.

Evaluation of the Antimicrobial Potential and Characterization of Novel T7-Like Erwinia Bacteriophages.

The recent outbreak of blight in pome fruit plants has been a major concern as there are two indistinguishable Erwinia species, Erwinia amylovora and E. pyrifoliae, which cause blight in South Korea. Although there is a strict management protocol consisting of antibiotic-based prevention, the area and the number of cases of outbreaks have increased. In this study, we isolated four bacteriophages, pEp_SNUABM_03, 04, 11, and 12, that infect both E. amylovora and E. pyrifoliae and evaluated their potential as antimicrobial agents for administration against Erwinia-originated blight in South Korea. Morphological analysis revealed that all phages had podovirus-like capsids. The phage cocktail showed a broad spectrum of infectivity, infecting 98.91% of E. amylovora and 100% of E. pyrifoliae strains. The antibacterial effect was observed after long-term cocktail treatment against E. amylovora, whereas it was observed for both short- and long-term treatments against E. pyrifoliae. Genomic analysis verified that the phages did not encode harmful genes such as antibiotic resistance or virulence genes. All phages were stable under general orchard conditions. Collectively, we provided basic data on the potential of phages as biocontrol agents that target both E. amylovora and E. pyrifoliae.

Phage Cocktail in Combination with Kasugamycin as a Potential Treatment for Fire Blight Caused by Erwinia amylovora.

Recently, there has been an increasing number of blight disease reports associated with Erwinia amylovora and Erwinia pyrifoliae in South Korea. Current management protocols that have been conducted with antibiotics have faced resistance problems and the outbreak has not decreased. Because of this concern, the present study aimed to provide an alternative method to control the invasive fire blight outbreak in the nation using bacteriophages (phages) in combination with an antibiotic agent (kasugamycin). Among 54 phage isolates, we selected five phages, pEa_SNUABM_27, 31, 32, 47, and 48, based on their bacteriolytic efficacy. Although only phage pEa_SNUABM_27 showed host specificity for E. amylovora, all five phages presented complementary lytic potential that improved the host infectivity coverage of each phage All the phages in the cocktail solution could lyse phage-resistant strains. These strains had a decreased tolerance to the antibiotic kasugamycin, and a synergistic effect of phages and antibiotics was demonstrated both in vitro and on immature wound-infected apples. It is noteworthy that the antibacterial effect of the phage cocktail or phage cocktail-sub-minimal inhibitory concentration (MIC) of kasugamycin was significantly higher than the kasugamycin at the MIC. The selected phages were experimentally stable under environmental factors such as thermal or pH stress. Genomic analysis revealed these are novel Erwinia-infecting phages, and did not encode antibiotic-, virulence-, or lysogenic phage-related genes. In conclusion, we suggest the potential of the phage cocktail and kasugamycin combination as an effective strategy that would minimize the use of antibiotics, which are being excessively used in order to control fire blight pathogens.

Isolation of Nine Bacteriophages Shown Effective against Erwinia amylovora in Korea.

Erwinia amylovora is a devastating bacterial plant pathogen that infects Rosaceae including apple and pear and causes fire blight. Bacteriophages have been considered as a biological control agent for preventing bacterial infections of plants. In this study, nine bacteriophages (ΦFifi011, ΦFifi044, ΦFifi051, ΦFifi067, ΦFifi106, ΦFifi287, ΦFifi318, ΦFifi450, and ΦFifi451) were isolated from soil and water samples in seven orchards with fire blight in Korea. The genetic diversity of bacteriophage isolates was confirmed through restriction fragment length polymorphism pattern analysis. Host range of the nine phages was tested against 45 E. amylovora strains and 14 E. pyrifoliae strains and nine other bacterial strains. Among the nine phages, ΦFifi044 and ΦFifi451 infected and lysed E. amylovora only. And the remaining seven phages infected both E. amylovora and E. pyrifoliae. The results suggest that the isolated phages were different from each other and effective to control E. amylovora, providing a basis to develop biological agents and utilizing phage cocktails.

Staphylococcus aureus Does Not Synthesize Arginine from Proline under Physiological Conditions.

The Gram-positive pathogen Staphylococcus aureus is the only bacterium known to synthesize arginine from proline via the arginine-proline interconversion pathway despite having genes for the well-conserved glutamate pathway. Since the proline-arginine interconversion pathway is repressed by CcpA-mediated carbon catabolite repression (CCR), CCR has been attributed to the arginine auxotrophy of S. aureus. Using ribose as a secondary carbon source, here, we demonstrate that S. aureus arginine auxotrophy is not due to CCR but due to the inadequate concentration of proline degradation product. Proline is degraded by proline dehydrogenase (PutA) into pyrroline-5-carboxylate (P5C). Although the PutA expression was fully induced by ribose, the P5C concentration remained insufficient to support arginine synthesis because P5C was constantly consumed by the P5C reductase ProC. When the P5C concentration was artificially increased by either PutA overexpression or proC deletion, S. aureus could synthesize arginine from proline regardless of carbon source. In contrast, when the P5C concentration was reduced by overexpression of proC, it inhibited the growth of the ccpA deletion mutant without arginine. Intriguingly, the ectopic expression of the glutamate pathway enzymes converted S. aureus into arginine prototroph. In an animal experiment, the arginine-proline interconversion pathway was not required for the survival of S. aureus. Based on these results, we concluded that S. aureus does not synthesize arginine from proline under physiological conditions. We also propose that arginine auxotrophy of S. aureus is not due to the CcpA-mediated CCR but due to the inactivity of the conserved glutamate pathway. IMPORTANCE Staphylococcus aureus is a versatile Gram-positive human pathogen infecting various human organs. The bacterium's versatility is partly due to efficient metabolic regulation via the carbon catabolite repression system (CCR). S. aureus is known to interconvert proline and arginine, and CCR represses the synthesis of both amino acids. However, when CCR is released by a nonpreferred carbon source, S. aureus can synthesize proline but not arginine. In this study, we show that, in S. aureus, the intracellular concentration of pyrroline-5-carboxylate (P5C), the degradation product of proline and the substrate of proline synthesis, is too low to synthesize arginine from proline. These results call into question the notion that S. aureus synthesizes arginine from proline.

The Bacteriophage pEp_SNUABM_08 Is a Novel Singleton Siphovirus with High Host Specificity for Erwinia pyrifoliae.

Species belonging to the genus Erwinia are predominantly plant pathogens. A number of bacteriophages capable of infecting Erwinia have been used for the control of plant diseases such as fire blight. Public repositories provide the complete genome information for such phages, which includes genomes ranging from 30 kb to 350 kb in size. However, limited information is available regarding bacteriophages belonging to the family Siphoviridae. A novel lytic siphophage, pEp_SNUABM_08, which specifically infects Erwinia pyrifoliae, was isolated from the soil of an affected apple orchard in South Korea. A comprehensive genome analysis was performed using the Erwinia-infecting siphophage. The whole genome of pEp_SNUABM_08 comprised 62,784 bp (GC content, 57.24%) with 79 open reading frames. The genomic characteristics confirmed that pEp_SNUABM_08 is a singleton lytic bacteriophage belonging to the family Siphoviridae, and no closely related phages have been reported thus far. Our study not only characterized a unique phage, but also provides insight into the genetic diversity of Erwinia bacteriophages.

Characterization of Novel Erwinia amylovora Jumbo Bacteriophages from Eneladusvirus Genus.

Jumbo phages, which have a genome size of more than 200 kb, have recently been reported for the first time. However, limited information is available regarding their characteristics because few jumbo phages have been isolated. Therefore, in this study, we aimed to isolate and characterize other jumbo phages. We performed comparative genomic analysis of three Erwinia phages (pEa_SNUABM_12, pEa_SNUABM_47, and pEa_SNUABM_50), each of which had a genome size of approximately 360 kb (32.5% GC content). These phages were predicted to harbor 546, 540, and 540 open reading frames with 32, 34, and 35 tRNAs, respectively. Almost all of the genes in these phages could not be functionally annotated but showed high sequence similarity with genes encoded in Serratia phage BF, a member of Eneladusvirus. The detailed comparative and phylogenetic analyses presented in this study contribute to our understanding of the diversity and evolution of Erwinia phage and the genus Eneladusvirus.

Purification and Characterization of Pasteuricin Produced by Staphylococcus pasteuri RSP-1 and Active against Multidrug-Resistant Staphylococcus aureus.

Staphylococcus aureus is an important pathogen implicated in various diseases, including staphylococcal food poisoning. Bacteriocins are considered safe and effective antimicrobial substances for the prevention of the growth of pathogenic bacteria. In this article, we describe the purification and characterization of pasteuricin, a novel bacteriocin produced by Staphylococcus pasteuri RSP-1. A cell-free supernatant of S. pasteuri RSP-1 exerted strong antimicrobial activity against staphylococci, including methicillin-resistant S. aureus (MRSA), and gram-positive bacteria. The loss of antimicrobial activity upon treatment with proteolytic enzymes confirmed the proteinaceous nature of pasteuricin. A rapid and pronounced bactericidal effect of pasteuricin was confirmed by a live-dead bacterial viability assay. To our knowledge, pasteuricin is the first reported S. pasteuri bacteriocin that inhibits S. aureus. Because pasteuricin is characterized by strong antimicrobial activity and high stability, it has potential as an alternative antimicrobial agent to antibiotics.

Improved multiplex PCR primers for rapid identification of coagulase-negative staphylococci.

Coagulase-negative staphylococci (CNS) are opportunistic pathogens that are currently emerging as causative agents of human disease. Though CNS are widespread in the clinic and food, their precise identification at species level is important. Here, using 16S rRNA sequencing, 55 staphylococcal isolates were identified as S. capitis, S. caprae, S. epidermidis, S. haemolyticus, S. pasteuri, S. saprophyticus, S. warneri, and S. xylosus. Although 16S rRNA sequencing is universally accepted as a standard for bacterial identification, the method did not effectively discriminate closely related species, and additional DNA sequencing was required. The divergence of the sodA gene sequence is higher than that of 16S rRNA. To devise a rapid and accurate identification method, sodA-specific primers were designed to demonstrate that species-specific multiplex polymerase chain reaction (PCR) can be used for the identification of CNS species. The accuracy of this method was higher than that of phenotypic identification; the method is simple and less time-consuming than 16S rRNA sequencing. Of the 55 CNS isolates, 92.72% were resistant to at least one antibiotic, and 60% were resistant to three or more antibiotics. CNS isolates produced diverse virulence-associated enzymes, including hemolysin (produced by 69.09% of the isolates), protease (65.45%), lipase (54.54%), lecithinase (36.36%), and DNase (29.09%); all isolates could form a biofilm. Because of the increasing pathogenic significance of CNS, the efficient multiplex PCR detection method developed in this study may contribute to studies for human health.

Multiplexed Detection of Foodborne Pathogens from Contaminated Lettuces Using a Handheld Multistep Lateral Flow Assay Device.

This paper presents a handheld device that is capable of simplifying multistep assays to perform sensitive detection of foodborne pathogens. The device is capable of multiplexed detection of Escherichia coli (E. coli) O157:H7, Salmonella Typhimurium (S. Typhimurium), Staphylococcus aureus, and Bacillus cereus. The limit of detection for each bacterium was characterized, and then, the detection of bacteria from contaminated fresh lettuces was demonstrated for two representative foodborne pathogens. We employed a sample pretreatment protocol to recover and concentrate target bacteria from contaminated lettuces, which can detect 1.87 × 104 CFU of E. coli O157:H7 and 1.47 × 104 CFU of S. Typhimurium/1 g of lettuce without an enrichment process. Lastly, we demonstrated that the limit of detection can be reduced to 1 CFU of E. coli O157:H7 and 1 CFU of S. Typhimurium/1 g of lettuce by including a 6 h enrichment of contaminated lettuces in growth media before pretreatment.

Genomic characterization of bacteriophage vB_PcaP_PP2 infecting Pectobacterium carotovorum subsp. carotovorum, a new member of a proposed genus in the subfamily Autographivirinae.

Bacteriophage vB_PcaP_PP2 (PP2) is a novel virulent phage that infects the plant-pathogenic bacterium Pectobacterium carotovorum subsp. carotovorum. PP2 phage has a 41,841-bp double-stranded DNA encoding 47 proteins, and it was identified as a member of the family Podoviridae by transmission electron microscopy. Nineteen of its open reading frames (ORFs) show homology to functional proteins, and 28 ORFs have been characterized as hypothetical proteins. PP2 phage is homologous to Cronobacter phage vB_CskP_GAP227 and Dev-CD-23823. Based on phylogenetic analysis, PP2 and its homologous bacteriophages form a new group within the subfamily Autographivirinae in the family Podoviridae, suggesting the need to establish a new genus. No lysogenic-cycle-related genes or bacterial toxins were identified.

Complete genome sequence analysis of a novel Staphylococcus phage StAP1 and proposal of a new species in the genus Silviavirus.

Bacteriophage StAP1 was isolated from a soil sample infecting Staphylococcus aureus and S. xylosus. Its genome was found to be 135,502 base pairs (bp) long with 30.00 mol% G+C content and 192 open reading frames. While no tRNA encoding genes were identified, 7 mobile elements were found to interrupt five StAP1 open reading frames. Comparative genomic and proteomic analysis consistently supports the establishment of a new species in the genus Silviavirus.

OmpF of Pectobacterium carotovorum subsp. carotovorum Pcc3 is required for carocin D sensitivity.

Carocin D is a bacteriocin produced by Pectobacterium carotovorum subsp. carotovorum Pcc21. Carocin D inhibits the growth of P carotovorum subsp. carotovorum and closely related strains. Pectobacterium carotovorum subsp. carotovorum is a causative bacterium for soft rot disease and leads to severe economic losses. Bacteriocins recognize and interact with a specific membrane protein of target bacteria as a receptor. To identify the receptor responsible for carocin D recognition, mutants that underwent a phenotypic change from carocin D sensitivity to carocin D insensitivity were screened. Based on Tn5 insertions, carocin D sensitivity was dependent on expression of the outer membrane protein OmpF. The insensitivity of the mutant (Pcc3MR) to carocin D was complemented with ompF from carocin D-sensitive strains, not from carocin D-resistant strains. The selectivity between sensitive and resistant strains could be attributed to variation in OmpFs in the cell-surface-exposed regions. Based on sequence analysis and complementation assays, it appears that carocin D uses OmpF as a receptor and is translocated by the TonB system. According to previously reported translocation mechanisms of colicins, OmpF works along with the TolA system rather than the TonB system. Therefore, the current findings suggest that carocin D is imported by a unique colicin-like bacteriocin translocation system.

Complete Genome Sequence of Biofilm-Forming Strain Staphylococcus haemolyticus S167.

Staphylococcus haemolyticus S167 has the ability to produce biofilms in large quantities. Genomic analyses revealed information on the biofilm-related genes of S. haemolyticus S167. Detailed studies of biofilm formation at the molecular level could provide a foundation for biofilm control research.

Genetic Diversity and Antibiotic Resistance Patterns of Staphylococcus Aureus Isolated from Leaf Vegetables in Korea.

Staphylococcus aureus is an important foodborne pathogen on global basis. The current study investigated the genetic patterns in S. aureus isolates from leaf vegetables (n = 53). Additional isolates from livestock (n = 31) and humans (n = 27) were compared with the leaf vegetable isolates. Genes associated with toxins, antibiotic resistance, and pulsed-field gel electrophoresis (PFGE) patterns were analyzed. At least 1 enterotoxin-encoding gene (sea, seb, sec, sed, and see) was detected in 11 of 53 (20.75%) leaf vegetable isolates. When the agr (accessory gene regulator) grouping was analyzed, agr II was the major group, whereas agr IV was not present in leaf vegetable isolates. All S. aureus isolates from leaf vegetables were resistant to more than one of the antibiotics tested. Nineteen of 53 (35.85%) isolates from leaf vegetables exhibited multidrug-resistance, and 11 of these were MRSA (methicillin-resistant S. aureus). A dendrogram displaying the composite types of S. aureus isolates from 3 origins was generated based on the combination of the toxin genes, agr genes, antibiotic resistance, and PFGE patterns. The isolates could be clustered into 8 major composite types. The genetic patterns of S. aureus isolates from leaf vegetables and humans were similar, whereas those from livestock had unique patterns. This suggests some S. aureus isolates from leaf vegetables to be of human origin.

Characterization and genomic analysis of two Staphylococcus aureus bacteriophages isolated from poultry/livestock farms.

Staphylococcus aureus is one of the most important pathogens, causing various diseases in humans and animals. As methicillin-resistant S. aureus (MRSA) has become increasingly prevalent, controlling this pathogen with standard antibiotic treatment has become challenging. Bacteriophages (phages) have attracted interest as alternative antibacterial agents to control MRSA. In this study, we isolated six S. aureus phages from soils of poultry/livestock farms. Based on the results of host range determination with 150 S. aureus strains and restriction enzyme treatment of phage DNA, two phages, designated SP5 and SP6, were selected for further characterization and genome sequencing. Both SP5 and SP6 were classified as members of the family Siphoviridae. The genome of SP5 comprises 43 305 bp and contains 63 ORFs, while the SP6 genome comprises 42 902 bp and contains 61 ORFs. Although they have different host spectra, the phage genomes exhibit high nucleotide similarity to each other. Adsorption assay results suggested that the host range determinants of the two phages are involved in both adsorption and infection. Comparative genomic analyses of the two phages provided evidence that the lysogenic/lytic control module and tail proteins may be important for host specificity.