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The isolation and identification of pathogenic bacteria from a variety of samples are critical for controlling bacterial infection-related health problems. The conventional methods, such as plate counting and polymerase chain reaction-based approaches, tend to be time-consuming and reliant on specific instruments, severely limiting the effective identification of these pathogens. In this study, we employed the specificity of the cell wall-binding (CBD) domain of the Staphylococcus aureus bacteriophage 80 alpha (80α) endolysin towards the host bacteria for isolation. Amidase 3-CBD conjugated magnetic beads successfully isolated as few as 1 × 10 2  CFU/mL of S. aureus cells from milk, blood, and saliva. The cell wall hydrolyzing activity of 80α endolysin promoted the genomic DNA extraction efficiency by 12.7 folds on average, compared to the commercial bacterial genomic DNA extraction kit. Then, recombinase polymerase amplification (RPA) was exploited to amplify the nuc gene of S. aureus from the extracted DNA at 37 °C for 30 min. The RPA product activated Cas12a endonuclease activity to cleave fluorescently labeled ssDNA probes. We then converted the generated signal into a fluorescent readout, detectable by either the naked eye or a portable, self-assembled instrument with ultrasensitivity. The entire procedure, from isolation to identification, can be completed within 2 h. The simplicity and sensitivity of the method developed in this study make it of great application value in S. aureus detection, especially in areas with limited resource supply., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objective: This study aimed to introduce a lytic bacteriophage against Staphylococcus saprophyticus from wastewater in Gorgan, northern Iran., Results: The vB_SsapS-46 phage was isolated from urban wastewater and formed round and clear plaques on bacterial culture. It was visualized by electron microscopy and had a large head (approximately 106 nm) and a long tail (approximately 150 nm), indicating that it belongs to the Siphoviridae family. The host range of vB_SsapS-46 was determined using a spot test on 35 S. saprophyticus clinical isolates, and it was able to lyse 12 of the 35 clinical isolates (34%). Finally, the relationship between phage sensitivity and adherence genes was assessed, revealing no significant correlation between phage sensitivity and the frequency of adherence genes. The vB_SsapS-46 phage can be used alone or in a mixture in future studies to control urinary tract infections caused by this bacterium, especially in the elimination of drug-resistant pathogens., (© 2024. The Author(s).)

Nowadays finding the new antimicrobials is necessary due to the emerging of multidrug resistant strains. The present study aimed to isolate and characterize bacteriophages against S. aureus. Strains Huma and Simurgh were the two podovirus morphology phages which isolated and then characterized. Huma and Simurgh had a genome size of 16,853 and 17,245 bp, respectively and both were Rosenblumvirus with G + C content of 29%. No lysogeny-related genes, nor virulence genes were identified in their genomes. They were lytic only against two out of four S. aureus strains. They also were able to inhibit S. aureus for 8 h in-vitro. Both showed a rapid adsorption. Huma and Simurgh had the latent period of 80 and 60 m and the burst sizes of 45 and 40 PFU/ml and also, they showed very low cell toxicity of 1.23%-1.79% on HT-29 cells, respectively. Thus, they can be considered potential candidates for biocontrol applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Staphylococcus aureus can be a harmless coloniser, but it can also cause severe infections in humans, livestock and wildlife. Regarding the latter, only few studies have been performed and knowledge on virulence factors is insufficient. The aim of the present study was to study S. aureus isolates from deceased wild beavers (Castor fiber). Seventeen isolates from eleven beavers, found in Germany and Austria, were investigated. Antimicrobial and biocide susceptibility tests were performed. Isolates were characterised using S. aureus-specific DNA microarrays, spa typing and whole-genome sequencing. From two isolates, prophages were induced by mitomycin C and studied by transmission electron microscopy. Four isolates belonged to clonal complex (CC) 8, CC12, and CC398. Twelve isolates belonged to CC1956 and one isolate was CC49. The CC49 and CC1956 isolates carried distinct lukF/S genes related to the Panton-Valentine leukocidin (PVL) from human isolates of S. aureus. These genes were located on related, but not identical, Siphovirus prophages. The beavers, from which those isolates originated, suffered from abscesses, purulent organ lesions and necrotising pneumonia, i.e., clinical manifestations resembling symptoms of severe PVL-associated disease in humans. It might thus be assumed that the "Beaver Leukocidin (BVL, lukF/S-BV)"-positive strains are beaver-specific pathogens, and further studies on their clinical role as well as on a possible transmissibility to other species, including humans, are warranted., (© 2021. The Author(s).)

The host range of bacteriophages defines their impact on bacterial communities and genome diversity. Here, we characterize 94 novel staphylococcal phages from wastewater and establish their host range on a diversified panel of 117 staphylococci from 29 species. Using this high-resolution phage-bacteria interaction matrix, we unveil a multi-species host range as a dominant trait of the isolated staphylococcal phages. Phage genome sequencing shows this pattern to prevail irrespective of taxonomy. Network analysis between phage-infected bacteria reveals that hosts from multiple species, ecosystems, and drug-resistance phenotypes share numerous phages. Lastly, we show that phages throughout this network can package foreign genetic material enclosing an antibiotic resistance marker at various frequencies. Our findings indicate a weak host specialism of the tested phages, and therefore their potential to promote horizontal gene transfer in this environment., (© 2021. The Author(s).)

The majority of previously described Staphylococcus aureus bacteriophages belong to three major groups, namely, P68-like podophages, Twort-like or K-like myophages, and a more diverse group of temperate siphophages. Here, we present the following three novel S. aureus "jumbo" phages: MarsHill, Madawaska, and Machias. These phages were isolated from swine production environments in the United States and represent a novel clade of S. aureus myophage. The average genome size for these phages is ∼269 kb with each genome encoding ∼263 predicted protein-coding genes. Phage genome organization and content are similar to those of known jumbo phages of Bacillus sp., including AR9 and vB_BpuM-BpSp. All three phages possess genes encoding complete virion and nonvirion RNA polymerases, multiple homing endonucleases, and a retron-like reverse transcriptase. Like AR9, all of these phages are presumed to have uracil-substituted DNA which interferes with DNA sequencing. These phages are also able to transduce host plasmids, which is significant as these phages were found circulating in swine production environments and can also infect human S. aureus isolates. IMPORTANCE This study describes the comparative genomics of the following three novel S. aureus jumbo phages: MarsHill, Madawaska, and Machias. These three S. aureus myophages represent an emerging class of S. aureus phage. These genomes contain abundant introns which show a pattern consistent with repeated acquisition rather than vertical inheritance, suggesting intron acquisition and loss are active processes in the evolution of these phages. These phages have presumably hypermodified DNA which inhibits sequencing by several different common platforms. Therefore, these phages also represent potential genomic diversity that has been missed due to the limitations of standard sequencing techniques. In particular, such hypermodified genomes may be missed by metagenomic studies due to their resistance to standard sequencing techniques. Phage MarsHill was found to be able to transduce host DNA at levels comparable to that found for other transducing S. aureus phages, making it a potential vector for horizontal gene transfer in the environment.

Staphylococcus aureus infections are of growing concern given the increased incidence of antibiotic resistant strains. Egypt, like several other countries, has seen alarming increases in methicillin-resistant S. aureus (MRSA) infections. This species can rapidly acquire genes associated with resistance, as well as virulence factors, through mobile genetic elements, including phages. Recently, we sequenced 56 S. aureus genomes from Alexandria Main University Hospital in Alexandria, Egypt, complementing 17 S. aureus genomes publicly available from other sites in Egypt. In the current study, we found that the majority (73.6%) of these strains contain intact prophages, including Biseptimaviruses, Phietaviruses, and Triaviruses. Further investigation of these prophages revealed evidence of horizontal exchange of the integrase for two of the prophages. These Egyptian S. aureus prophages are predicted to encode numerous virulence factors, including genes associated with immune evasion and toxins, including the Panton-Valentine leukocidin (PVL)-associated genes lukF -PV/ lukS -PV. Thus, prophages are likely to be a major contributor to the virulence of S. aureus strains in circulation in Egypt.

Background: Staphylococcus aureus is the causative agent of chronic mastitis, and can form a biofilm that is difficult to completely remove once formed. Disinfectants are effective against S. aureus, but their activity is easily affected by environmental factors and they are corrosive to equipment and chemically toxic to livestock and humans. Therefore, we investigated the potential utility of a bacteriophage as a narrow-spectrum disinfectant against biofilms formed by S. aureus. In this study, we isolated and characterized bacteriophage vB_SauM_SDQ (abbreviated to SDQ) to determine its efficacy in removing S. aureus biofilms., Results: SDQ belongs to the family Myoviridae and consists of a hexagonal head, long neck, and short tail. This phage can sterilize a 10 9  CFU/mL culture of S. aureus in 12 h and multiply itself 1000-fold in that time. Biofilms formed on polystyrene, milk, and mammary-gland tissue were significantly reduced after SDQ treatment. Fluorescence microscopy and scanning electron microscopy showed that SDQ destroyed the biofilm structure. Moreover, the titer of SDQ remained relatively high after the lysis of the bacteria and the removal of the biofilm, exerting a continuous bacteriostatic effect. SDQ also retained its full activity under conditions that mimic common environments, i.e., in the presence of nonionic detergents, tap water, or organic materials. A nonionic detergent (Triton X-100) enhanced the removal of biofilm by SDQ., Conclusions: Our results suggest that SDQ, a specific lytic S. aureus phage, can be used to control biofilm infections. SDQ maintains its full activity in the presence of nonionic detergents, tap water, metal chelators, and organic materials, and can be used in combination with detergents. We propose this phage as a narrow-spectrum disinfectant against S. aureus, to augment or supplement the use of broad-spectrum disinfectants in the prevention and control of the mastitis and dairy industry contamination caused by S. aureus.

Two Staphylococcus aureus bacteriophages, KSAP7 and KSAP11, were isolated from sewage and characterized. Based on morphology and DNA sequences, they were assigned to the genus Silviavirus, subfamily Twortvirinae, family Herelleviridae, whose members are hypothesized to be suitable for bacteriophage therapy. The KSAP7 and KSAP11 genomes were 137,950 and 138,307 bp in size, respectively. Although their DNA sequences were almost identical, evidence of site-specific DNA rearrangements was found in two regions. Changes in the number of PIEPEK amino acid sequence repeats encoded by orf10 and the insertion/deletion of a 541-bp sequence that includes a possible tail-related gene were identified.

In this study, the genome of a new strain of lytic Staphylococcus aureus Herelleviridae, vBSM-A1, was characterized and annotated. The phage was isolated from sewage samples collected in Xinjiang Province, China. The genome of vBSM-A1 was found to comprise a linear double-stranded DNA of 140,654 bp length, with a G + C content of 30.33%. A total of 215 ORFs were detected in the phage DNA, 74 of which were functionally assigned. The 3D structure model of endolysin LysK (ORF 143) was created using Phyre2.

Staphylococcus aureus is a notorious foodborne pathogen since it has ability to produce variety of toxins including heat-stable enterotoxin, form biofilm, and acquire resistance to antibiotics. Biocontrol of foodborne pathogens by lytic bacteriophages garners increasing interest from both researchers and food industry. In the present study, 29 phages against S. aureus were successfully isolated from chicken, pork, and fish. Characterization of the isolates revealed that phage SA46-CTH2 belonging to Podoviridae family had a number of features suitable for food industry applications such as wide host range, short latent period, large burst size, high stress tolerance, and a genome free of virulence genes. Furthermore, phage SA46-CTH2 alone or in combination with nisin exhibited great efficacy in reducing planktonic and biofilm cells of S. aureus at various conditions tested. The combination of phage SA46-CTH2 and nisin was also found to be able to inhibit the regrowth of S. aureus at both 37 and 24 °C.Key points• A total of 29 S. aureus phages were successfully isolated from fish, pork, and chicken products. • Phage SA46-CTH2 was characterized by host range, morphology, and genome sequencing. • SA46-CTH2 significantly reduced both planktonic and biofilm cells of S. aureus. • Combination of SA46-CTH2 and nisin inhibited the regrowth of S. aureus.

Brazil has the second-largest dairy cattle herd in the world, and bovine mastitis still can cause significant losses for dairy farmers. Despite this fact, little information is available about milk microbial composition of Brazilian dairy cows, as well as the potential use of bacteriophages in the control of S. aureus. Here, we investigated milk bacterial composition of 28 Holstein Fresian cows (109 teats), selected in the dry-off period, using 16S rRNA analysis. Furthermore, a representative S. aureus strain (UFV2030RH1) was obtained at drying-off for isolation of a bacteriophage (vB_SauM-UFV_DC4, UFV_DC4) and bacterial genomic comparison purposes. Our outcomes revealed that Staphylococcus was the third most prevalent genus and positively correlated with subclinical mastitis events. As a major finding, genomic analyses showed the presence of adhesive matrix molecules that recognize microbial surface components (MSCRAMM) in UFV2030RH1 and might indicate great biofilm formation capability. A minimum inhibitory concentration (MIC) assay showed that resistance to ampicillin was the highest among the antibiotic tested in S. aureus 3059 and UFV2030RH1, displaying values four and sixteen times greater than MIC resistance breakpoint, respectively. Together, our results suggest that Staphylococcus is highly prevalent in dairy cows at drying-off and the use of the phage UFV_DC4 as a biocontrol agent must be investigated in future studies.

The emergence of life-threatening methicillin-resistant Staphylococcus aureus (MRSA) has led to increased interest in the use of bacteriophages as an alternative therapy to antibiotics. The success of phage therapy is greatly dependent on the selected phage possessing a wide host range. This study describes phage ɸMR003 isolated from sewage influent at a municipal wastewater treatment plant in Tokyo, Japan. ɸMR003 could infect 97% of 104 healthcare- and community-associated MRSA strains tested, compared with 73% for phage ɸSA012, which has a broad host range against bovine mastitis S. aureus. Genome analysis revealed that ɸMR003 belongs to the genus Silviavirus which has not been studied extensively. ɸMR003 recognizes and binds to wall teichoic acid (WTA) of S. aureus during infection. In silico comparisons of the genomes of ɸMR003 and ɸSA012 revealed that ORF117 and ORF119 of ɸMR003 are homologous to the putative receptor-binding proteins ORF103 and ORF105 of ɸSA012, with amino acid similarities of 75% and 72%, respectively. ORF104, which is an N-acetylglucosaminidase found in the ɸMR003 tail, may facilitate phage's infection onto the WTA-null S. aureus RN4220. The differences in tail and baseplate proteins may be key contributing factors to the different host specificities of ɸMR003 and ɸSA012. ɸMR003 showed strong adsorptivity, but not infectivity, against S. aureus SA003, which may be influenced by the bacterium's restriction modification system. This study expands our knowledge of the genomic diversity and host specificity of Silviavirus, which is a potential phage therapy candidate for MRSA infections.

A potential concern with bacteriophage (phage) therapeutics is a host-versus-phage response in which the immune system may neutralize or destroy phage particles and thus impair therapeutic efficacy, or a strong inflammatory response to repeated phage exposure might endanger the patient. Current literature is discrepant with regard to the nature and magnitude of innate and adaptive immune response to phages. The purpose of this work was to study the potential effects of Staphylococcus aureus phage K on the activation of human monocyte-derived dendritic cells. Since phage K acquired from ATCC was isolated around 90 years ago, we first tested its activity against a panel of 36 diverse S. aureus clinical isolates from military patients and found that it was lytic against 30/36 (83%) of strains. Human monocyte-derived dendritic cells were used to test for an in vitro phage-specific inflammatory response. Repeated experiments demonstrated that phage K had little impact on the expression of pro- and anti-inflammatory cytokines, or on MHC-I/II and CD80/CD86 protein expression. Given that dendritic cells are potent antigen-presenting cells and messengers between the innate and the adaptive immune systems, our results suggest that phage K does not independently affect cellular immunity or has a very limited impact on it.

The bacteriophage K1/420 is a member of genus Kayvirus that was extensively studied as an alternative treatment to combat bacterial infections caused by antibiotic-resistant Staphylococcus aureus strains. Despite the promise of phage therapy, the development of clinical applications of phages is facing regulatory and technical hurdles before it can receive acceptance in the Western World. Suitable simple and accurate diagnostic techniques to control the quality of the phage, which would satisfy the requirements of regulatory authorities are still being discussed. Here, we present the conditions for the simultaneous separation and detection of phage K1/420 and S. aureus by CZE and by CIEF were found, and the phage isoelectric point was determined to be 3.6. After removing the cell debris, the phage was successfully purified from the crude phage lysate and pre-concentrated by preparative isoelectric focusing. Its zone was localized by the positions of colored pI markers in the cellulose bed. The phage from the harvested zone had a decreased ability to infect its host. However, it was suitable for its separation, detection and identification by capillary electrophoretic methods, MALDI-TOF MS and electron microscopy., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Staphylococcus aureus is a commensal and pathogenic bacterium that causes infections in humans and animals. It is a major cause of nosocomial infections worldwide. Due to increasing prevalence of multidrug resistance, alternative methods to eradicate the pathogen are necessary. In this respect, polyvalent staphylococcal myoviruses have been demonstrated to be excellent candidates for phage therapy. Here we present the characterization of the bacteriophage vB_SauM-fRuSau02 (fRuSau02) that was isolated from a commercial Staphylococcus bacteriophage cocktail produced by Microgen (Moscow, Russia). The genomic analysis revealed that fRuSau02 is very closely related to the phage MSA6, and possesses a large genome (148,464 bp), with typical modular organization and a low G+C (30.22%) content. It can therefore be classified as a new virus among the genus Twortlikevirus . The genome contains 236 predicted genes, 4 of which were interrupted by insertion sequences. Altogether, 78 different structural and virion-associated proteins were identified from purified phage particles by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The host range of fRuSau02 was tested with 135 strains, including 51 and 54 Staphylococcus aureus isolates from humans and pigs, respectively, and 30 coagulase-negative Staphylococcus strains of human origin. All clinical S. aureus strains were at least moderately sensitive to the phage, while only 39% of the pig strains were infected. Also, some strains of Staphylococcus intermedius , Staphylococcus lugdunensis , Staphylococcus epidermidis , Staphylococcus haemolyticus , Staphylococcus saprophyticus and Staphylococcus pseudointer were sensitive. We conclude that fRuSau02, a phage therapy agent in Russia, can serve as an alternative to antibiotic therapy against S. aureus ., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

A Staphylococcus aureus (S. aureus)-specific lytic bacteriophage P-S. aureus-9, isolated from an environmental water sample, was assembled on magnetic beads for capturing S. aureus from samples through magnetic separation. Horseradish Peroxidase (HRP) labeled immunoglobulin (IgG) antibodies were used to detect the captured S. aureus by reacting with protein A on S. aureus followed by colorimetric signals, which were generated from the catalytic reaction between HRP and the substrate 3,3',5,5'-Tetramethylbenzidine (TMB). Under optimal conditions, the calibration curve was linear from 1.0×10 4 to 1.0×10 6 CFUmL -1 . The limit of detection (LOD) for the assay was 2.47×10 3 CFUmL -1 and 8.86×10 3 CFUmL -1 of S. aureus in PBS and apple juice, respectively. Moreover, the whole assay revealed outstanding specificity towards S. aureus, without any interference of common pathogenic bacteria, and can be completed within 90min without any pre-enrichment. As far as known, it was the first time to detect S. aureus based on the double site recognition of bacteriophage and mammal IgG. The novel approach has shown good potentials for a rapid, specific, cheap and simple detection of S. aureus in food samples., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: The implementation of phage therapy is re-emerging with the increase in widespread antibiotic-resistant bacteria., Methods: Staphylococcus phage JD007 was characterized and its complete genome sequence analysed., Results: Staphylococcus phage JD007 was classified as belonging to the Myoviridae family based on its morphology, as observed by transmission electron microscopy. Its lytic activity was stable between pH 5-11 and below 42 °C; moreover, an absorbance curve showed that nearly 90% of the viral particles had adsorbed to its host after a 20 min co-incubation. The complete genome size is 141,836 bp, making JD007 one of the largest Staphylococcus phages of Myoviridae. No identifiable resistance or virulence genes were found in the JD007 genome. JD007 was able to lyse 95% of S. aureus isolates, including the prevalent ST239-MRSA and ST59-MRSA strains isolated from different hospitals in Shanghai, China, and inhibition assays showed that JD007 could inhibit S. aureus growth at a multiplicity of infection of 0.1., Conclusions: The results suggested that Staphylococcus phage JD007 can potentially be used in phage therapy or for the detection of S. aureus.

Introduction: The use of bacteriophages as an alternative treatment method against multidrug-resistant bacteria has not been explored in Kenya. This study sought to determine the efficacy of environmentally obtained lytic bacteriophage against multidrug-resistant Staphylococcus aureus (MDRSA) bacterium in mice., Methodology: Staphylococcus aureus bacterium and S. aureus-specific lytic phage were isolated from sewage and wastewater collected within Nairobi County, Kenya. Thirty mice were randomly assigned into three groups: MDRSA infection group (n = 20), phage-infection group (n = 5), and non-infection group (n = 5). The MDRSA infection group was further subdivided into three groups: clindamycin treatment (8 mg/kg; n = 5), lytic phage treatment (108 PFU/mL (n = 5), and a combination treatment of clindamycin and lytic phage (n = 5). Treatments were done at either 24 or 72 hours post-infection (p.i), and data on efficacy, bacterial load, and animal physical health were collected., Results: Treatment with phage was more effective (100%) than with clindamycin (62.25% at 24 hours p.i and 87.5% at 72 hours p.i.) or combination treatment (75% at 24 hours p.i. and 90% at 72 hours p.i.) (p < 0.001)., Conclusions: The results show that the environmentally obtained S. aureus lytic bacteriophage has therapeutic potential against MDRSA bacterium in mice.

Temperate phages have been suggested to carry virulence factors and other lysogenic conversion genes that play important roles in pathogenicity. In this study, phage TEM123 in wild-type Staphylococcus aureus from food sources was analyzed with respect to its morphology, genome sequence, and antibiotic resistance conversion ability. Phage TEM123 from a mitomycin C-induced lysate of S. aureus was isolated from foods. Morphological analysis under a transmission electron microscope revealed that it belonged to the family Siphoviridae. The genome of phage TEM123 consisted of a double-stranded DNA of 43,786 bp with a G+C content of 34.06%. A bioinformatics analysis of the phage genome identified 43 putative open reading frames (ORFs). ORF1 encoded a protein that was nearly identical to the metallo-β-lactamase enzymes that degrade β-lactam antibiotics. After transduction to S. aureus with phage TEM123, the metallo-β-lactamase gene was confirmed in the transductant by PCR and sequencing analyses. In a β-lactam antibiotic susceptibility test, the transductant was more highly resistant to β-lactam antibiotics than S. aureus S133. Phage TEM123 might play a role in the transfer of β-lactam antibiotic resistance determinants in S. aureus. Therefore, we suggest that the prophage of S. aureus with its exotoxin is a risk factor for food safety in the food chain through lateral gene transfer.

Stau2 is a lytic myophage of Staphylococcus aureus isolated from medical specimen. Exhibiting a broad host range against S. aureus clinical isolates, Stau2 is potentially useful for topical phage therapy or as an additive in food preservation. In this study, Stau2 was firstly revealed to possess a circularly permuted linear genome of 133,798 bp, with low G + C content, containing 146 open reading frames, but encoding no tRNA. The genome is organized into several modules containing genes for packaging, structural proteins, replication/transcription and host-cell-lysis, with the structural proteins and DNA polymerase modules being organized similarly to that in Twort-like phages of Staphylococcus. With the encoded DNA replication genes, Stau2 can possibly use its own system for replication. In addition, analysis in silico found several introns in seven genes, including those involved in DNA metabolism, packaging, and structure, while one of them (helicase gene) is experimentally confirmed to undergo splicing. Furthermore, phylogenetic analysis suggested Stau2 to be most closely related to Staphylococcus phages SA11 and Remus, members of Twort-like phages. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis showed 14 structural proteins of Stau2 and N-terminal sequencing identified three of them. Importantly, this phage does not encode any proteins which are known or suspected to be involved in toxicity, pathogenicity, or antibiotic resistance. Therefore, further investigations of feasible therapeutic application of Stau2 are needed.

Methicillin-resistant Staphylococcus aureus (MRSA) is an increasing cause of serious infection, both in the community and hospital settings. Despite sophisticated strategies and efforts, the antibiotic options for treating MRSA infection are narrowing because of the limited number of newly developed antimicrobials. Here, four newly-isolated MRSA-virulent phages, IME-SA1, IMESA2, IME-SA118 and IME-SA119, were sequenced and analyzed. Their genome termini were identified using our previously proposed "termini analysis theory". We provide evidence that remarkable conserved terminus sequences are found in IME-SA1/2/118/119, and, moreover, are widespread throughout Twortlikevirus Staphylococcus phage G1 and K species. Results also suggested that each phage of the two species has conserved 5' terminus while the 3' terminus is variable. More importantly, a variable region with a specific pattern was found to be present near the conserved terminus of Twortlikevirus S. phage G1 species. The clone with the longest variable region had variable terminus lengths in successive generations, while the clones with the shortest variable region and with the average length variable region maintained the same terminal length as themselves during successive generations. IME-SA1 bacterial infection experiments showed that the variation is not derived from adaptation of the phage to different host strains. This is the first study of the conserved terminus and variable region of Twortlikevirus S. phages.

A novel bacteriophage that infects S. aureus, SA97, was isolated and characterized. The phage SA97 belongs to the Siphoviridae family, and the cell wall teichoic acid (WTA) was found to be a host receptor of the phage SA97. Genome analysis revealed that SA97 contains 40,592 bp of DNA encoding 54 predicted open reading frames (ORFs), and none of these genes were related to virulence or drug resistance. Although a few genes associated with lysogen formation were detected in the phage SA97 genome, the phage SA97 produced neither lysogen nor transductant in S. aureus. These results suggest that the phage SA97 may be a promising candidate for controlling S. aureus.

Exfoliative toxin A (ETA)-coding temperate bacteriophages are leading contributors to the toxic phenotype of impetigo strains of Staphylococcus aureus. Two distinct eta gene-positive bacteriophages isolated from S. aureus strains which recently caused massive outbreaks of pemphigus neonatorum in Czech maternity hospitals were characterized. The phages, designated ϕB166 and ϕB236, were able to transfer the eta gene into a prophageless S. aureus strain which afterwards converted into an ETA producer. Complete phage genome sequences were determined, and a comparative analysis of five designed genomic regions revealed major variances between them. They differed in the genome size, number of open reading frames, genome architecture, and virion protein patterns. Their high mutual sequence similarity was detected only in the terminal regions of the genome. When compared with the so far described eta phage genomes, noticeable differences were found. Thus, both phages represent two new lineages of as yet not characterized bacteriophages of the Siphoviridae family having impact on pathogenicity of impetigo strains of S. aureus.

A large collection of Staphylococcus aureus including a. 745 clinically significant isolates that were consecutively recovered from human infections during 2012-2013, b. 19 methicillin-susceptible (MSSA), randomly selected between 2006-2011 from our Staphylococcal Collection, c. 16 human colonizing isolates, and d. 10 strains from colonized animals was investigated for the presence and the molecular characteristics of CC398. The study was conducted in Thessaly, a rural region in Greece. The differentiation of livestock-associated clade from the human clade was based on canSNPs combined with the presence of the φ3 bacteriophage and the tetM, scn, sak, and chp genes. Among the 745 isolates, two MRSA (0.8% of total MRSA) and thirteen MSSA (2.65% of total MSSA) were found to belong to CC398, while, between MSSA of our Staphylococcal Collection, one CC398, isolated in 2010, was detected. One human individual, without prior contact with animals, was found to be colonized by a MSSA CC398. No CC398 was identified among the 10 S. aureus isolated from animals. Based on the molecular markers, the 17 CC398 strains were equally placed in the livestock-associated and in the human clades. This is the first report for the dissemination of S. aureus CC398 among humans in Greece.

Staphylococcus aureus is a primary pathogen that causes bovine mastitis resulting in serious economic losses and herd management problems in dairy cows. A novel bacteriophage, JS01, specifically infecting bovine S. aureus, was isolated from milk of mastitis-affected cattle. TEM observation showed that it belonged to the family Siphovirus. The JS01 strain demonstrated a broad host range. The prediction result of PHACTS suggested that the JS01 strain was temperate phage. The JS01 genome is 43,458 bp long, with a GC content of 33.32% and no tRNAs. Annotation and functional analysis of the predicted ORFs revealed six functional groups: structure and morphology, DNA replication and regulation, packaging, lysogeny, lysis, and pathogenicity. Comparative analysis between JS01, S. aureus MSSA476, and S. aureus prophage PVL was also performed. The characterization and genomic analysis of JS01 provide a better understanding of S. aureus-targeting bacteriophages and useful information for the development of phage-based biocontrol agents against S. aureus.

Using bacteriophages as a tool to the control of pathogens is a complementary to antibiotic therapy. We have isolated a lytic bacteriophage, designated vB&#95;SauM&#95;JS25, from sewage effluent on a dairy farm in Jiangsu, Mid-east of China for use as a biocontrol agent against Staphylococcus aureus infections. Phage vB&#95;SauM&#95;JS25 was morphologically classified as Myoviridae. The phage showed broad host ranges within S. aureus strains, lysing 51 of 56 strains (91.1 %). Its latent period and burst size were approximately 20 min and 21 PFU/cell, respectively. Phage vB&#95;SauM&#95;JS25 was able to survive in a pH range between 6 and 9. However, a treatment of 70 or 80 °C for 10 min completely inactivated the phage. Moreover, morphologic analysis of vB&#95;SauM&#95;JS25 revealed that it was closely related to other Myoviridae phages infecting Staphylococcus species. The bacteriolytic activity of phage vB&#95;SauM&#95;JS25 at a multiplicity infection (MOI) 1 indicted its efficiency for reducing bacterial growth. These findings suggest that phage vB&#95;SauM&#95;JS25 could be considered a potential therapeutic or prophylactic candidate against S. aureus infection.

Relatively few phages (<10) of coagulase negative staphylococci (CoNS) have been described. Staphylococcus epidermidis phage vB&#95;SepS&#95;SEP9 is a siphovirus with a unique morphology as a staphylococcal phage, possessing a very long tail. Its genome is unique and unrelated to any phage genomes deposited in public databases. It appears to encode a nonfunctional integrase. Due to the not having a recognizable lysogeny module, the phage is unable lysogenize. The genome comprises 129 coding sequences (CDS), 46 of which have an assigned function and 59 are unique. Its unique morphology and genome led to the proposal of the establishment of a new Siphoviridae genus named "Sep9likevirus"., (Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Mastitis in dairy cattle continues to be an economically important disease. However, control is complicated by a high prevalence of resistance to antibiotics. Phage therapy, therefore, is considered as an alternative way of controlling bacterial infections and contaminations. In this study, we have described isolation and characterization of a highly virulent phage SPW from wastewater of dairy farm, which possesses a strong lytic capability against mastitis-associated Staphylococcus aureus, the most important pathogen in bovine clinical and subclinical mastitis. The phage SPW produced large, round and clear plaques on bacterial culture plates. TEM showed phage SPW has an icosahedral head 62.5 nm in diameter and long tail of 106 nm, head and tail were held together by a connector of 18 ± 1.5 nm long and can be classified as a member of the Myoviridae family. Restriction analysis indicated that phage SPW was a dsDNA virus with an approximate genome size of 65-69 kb. One-step growth kinetics showed a short latency period of about 10-15 min and a rise period of 50 min and a relatively small burst size was 44 ± 3 phages particles/infected cell. Moreover, adsorption rates were not influenced by calcium ions and phage SPW was relatively stable in a wide range of temperature and pH values, and resistant to chloroform and isopropanol. The optimal multiplicity of infection (MOI) was 0.01. When phage SPW was used to infect five other clinically isolated pathogenic isolates, it showed relatively wide spectrum host range. Phage SPW was capable of eliciting efficient lysis of S. aureus, revealing it potentially as an effective approach to prophylaxis or treatment of S. aureus-associated mastitis in dairy cows.

The content of empirically selected bacteriophage mixtures, produced by Microgen for the prevention and treatment of staphylococcal and pseudomonade infections, was investigated by negative stain electron microscopy. The main population of phages was shown to belong to the groups suitable for therapeutic purposes based on bioinformatics analysis of known genomes of Pseudomonas and Staphylococcus phages. However, the phage morphology studies did not always reveal the exact correspondence of the phage to the exact group. Therefore, we suggest group genotyping of the therapeutic bacteriophages on thebasis of genetic conservative locus.

Staphylococcus aureus is a clinically important bacterium that is commensal in both humans and animals. Bacteriophage (phage) attachment to the host bacterial surface is an important process during phage infection, which involves interactions between phage receptor-binding proteins and host receptor molecules. However, little information is available on the receptor-binding protein of S. aureus phages. S. aureus virulent phages S24-1 and S13' (family Podoviridae, genus AHJD-like viruses) were isolated from sewage. In the present study, we investigated the receptor-binding protein of AHJD-like viruses using phage S24-1. First, based on a comparative genomic analysis of phages S24-1 and S13', open reading frame 16 (ORF16) of phage S24-1 was speculated to be the receptor-binding protein, which possibly determines the host range. Second, we demonstrated that this was the receptor-binding protein of phage S24-1. Third, our study suggested that wall teichoic acids in the cell walls of S. aureus are the main receptor molecules for ORF16 and phage S24-1. Finally, the C-terminal region of ORF16 may be essential for binding to S. aureus. These results strongly suggest that ORF16 of phage S24-1 and its homologs may be the receptor-binding proteins of AHJD-like viruses., (© 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Staphylococcus epidermidis is considered an important nosocomial pathogen, being very tolerant to the host immune system and antibiotherapy, particularly when in biofilms. Due to its high resistance, alternative antimicrobial strategies are under development. The use of bacteriophages is seen as an important strategy to combat pathogenic organisms. In this study, a S. epidermidis myovirus, SEP1, was isolated and characterized. The genome of this phage was sequenced and shown to be related peripherally to the genus Twortlikevirus. However, when compared with other phages of this genus, it showed DNA sequence identities no greater than 58.2 %. As opposed to other polyvalent viruses of the genus Twortlikevirus, SEP1 is highly specific to S. epidermidis strains. The good infectivity shown by this phage as well as its high lytic spectrum suggested that it might be a good candidate for therapeutic studies.

Some years ago, MRSA clonal complex (CC) 398 emerged, which spread extensively in livestock animals. People in contact with food production animals are at high risk of colonization. A reduction of MRSA CC398 in livestock might be achieved by application of virulent phages. However, there have not yet been any reports published on phages lysing MRSA CC398 strains. In this study, three virulent phages (PSa1, PSa2 and PSa3) with lytic activity against MRSA CC398 strains were isolated from German pig farms. Morphologically, the phages are members of the family Podoviridae, and they exhibited an identical host range. They lysed 52 (60 %) out of 86 tested MRSA CC398 strains representing 18 different spa types. While the PSa1 and PSa3 genomes have a similar size of approximately 17.5 kb, the PSa2 genome is somewhat larger (ca. 18.5 kb). Southern hybridization revealed strong DNA homologies between the phages, which was confirmed by sequence analysis of cloned restriction fragments and PCR products. Moreover, the whole PSa3 genomic sequence (17,602 bp) showed a close relationship to 44AHJD-like phages, which are not known to contain virulence-associated genes. To assess whether these phages might be candidates for applications, in vitro experiments were carried out in which the number of MRSA CC398 cells could be reduced by up to four log10 units. The phages were stable at a wide range of temperatures and pH values.

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.

Staphylococcus aureus is a well-known pathogen that causes several serious diseases in humans and animals. As part of the efforts to control this pathogen, a newly isolated bacteriophage, SA12, which specifically targets S. aureus, was characterized, and its genome was completely sequenced. Host range and bacteriophage challenge tests demonstrated its specific and efficient host lysis of S. aureus. The genome of phage SA12 consists of 42,902 bp length double-stranded DNA with 58 predicted ORFs-encoding phage structure, DNA manipulation, packaging, host lysis, and regulation proteins. The characterization and genome study of phage SA12 in this report is useful for understanding S. aureus-targeting bacteriophages and provides basic information for the further development of phage-based biocontrol agents against S. aureus.

From the four known isoforms of the staphylococcal exfoliative toxins (ETs), only ETA and ETB are the major causative agents. General knowledge is that the gene for ETA is located on the chromosome, whereas that for ETB is located on a large plasmid. Yoshizawa and co-workers (2000, Microbiol. Immunol. 44(3): 189-191) isolated, for the first time, a temperate phage (φETA) that carried the structural gene for ETA from an ETA-producing strain of Staphylococcus aureus. In this study, we presented eta gene encoding temperate phages isolated from methicillin-resistant S.aureus (MRSA) isolates obtained from patients in a Turkish hospital. Molecular analysis of the phage genome revealed that the eta gene is located upstream to amidase and holin genes, the same as in the φETA genome. However, partial sequence analysis of amidase and holin genes revealed polymorphic variation. In addition to polymorphic variation, restriction fragment length polymorphism (RFLP) analysis of all of the phage genomes showed that the ETA-containing phage is different from the rest of the phage genomes. The phylogenetic dendrogram of pulsed field gel electrophoresis (PFGE) analysis showed that the ETA-carrying MRSA is quite different from the rest of the MRSA strains. This is the first report showing that a MRSA strain carries an ETA-encoding phage.

A new siphophage (LH1) was isolated from raw milk using a Staphylococcus aureus ST352 host. Its genome (46,048 bp, 57 open reading frames) includes the two genes encoding Panton-Valentine leukocidin (PVL), a virulence factor usually harbored by S. aureus prophages. Nine structural proteins were identified, including a tail protein generated through a +1 frameshift. A phage lytic mutant was isolated, and its analysis revealed the deletion of genes coding for the PVL and an integrase. The deletion likely occurred through recombination between direct repeats.

Despite increasing interest in coagulase-negative staphylococci (CoNS), little information is available about their bacteriophages. We isolated and sequenced three novel temperate Siphoviridae phages (StB12, StB27, and StB20) from the CoNS Staphylococcus hominis and S. capitis species. The genome sizes are around 40 kb, and open reading frames (ORFs) are arranged in functional modules encoding lysogeny, DNA metabolism, morphology, and cell lysis. Bioinformatics analysis allowed us to assign a potential function to half of the predicted proteins. Structural elements were further identified by proteomic analysis of phage particles, and DNA-packaging mechanisms were determined. Interestingly, the three phages show identical integration sites within their host genomes. In addition to this experimental characterization, we propose a novel classification based on the analysis of 85 phage and prophage genomes, including 15 originating from CoNS. Our analysis established 9 distinct clusters and revealed close relationships between S. aureus and CoNS phages. Genes involved in DNA metabolism and lysis and potentially in phage-host interaction appear to be widespread, while structural genes tend to be cluster specific. Our findings support the notion of a possible reciprocal exchange of genes between phages originating from S. aureus and CoNS, which may be of crucial importance for pathogenesis in staphylococci.

A novel lytic bacteriophage, SA11, infecting Staphylococcus aureus was isolated, and the whole genome was sequenced. It belongs to the siphoviridae based on electron microscopic observation. It has a linear double-stranded DNA genome of 136,326 bp. Genomic analysis showed that it is distantly related to Staphylococcus phages A5W, K, ISP, Sb-1, and G1.

Methicillin-resistant Staphylococcus aureus (MRSA) from humans can be broadly separated into 3 groups: healthcare-associated (HA), community-associated (CA), and livestock-associated (LA) MRSA. Initially based on epidemiological features, division into these classes is becoming increasingly problematic. The sequencing of S. aureus genomes has highlighted variations in their accessory components, which likely account for differences in pathogenicity and epidemicity. In particular, temperate bacteriophages have been regarded as key players in bacterial pathogenesis. Bacteriophage-associated Panton-Valentine leukocidin genes (luk-PV) are regarded as epidemiological markers of the CA-MRSA due to their high prevalence in CA strains. This paper describes the development and application of a partial composite S. aureus virulence-associated gene microarray. Epidemic, pandemic, and sporadic lineages of UK HA and CA S. aureus were compared. Phage structural genes linked with CA isolates were identified and in silico analysis revealed these to be correlated with phage serogroup. CA strains predominantly carried a PVL-associated phage either of the A or Fb serogroup, whilst HA strains predominantly carried serogroup Fa or B phages. We speculate that carriage of a serogroup A/Fb PVL-associated phage rather than the luk-PV genes specifically is correlated with CA status., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

To determine the variation in the Panton-Valentine leukocidin (PVL) gene sequences and different PVL-encoding phages of Staphylococcus aureus strains collected from children in mainland China, fifty-eight strains with PVL collected from 2007 to 2009 were used. Their molecular characteristics were examined. Primers were designed to sequence the PVL genes. Six PVL-encoding phages (ϕPVL, ϕ108PVL, ϕSLT, ϕSa2MW, ϕSa2958, and ϕSa2USA) were identified by PCR. Eleven sequence types (ST) were detected with ST59 (39.7%, 23/58) the most frequent ST, followed by 910 (22.4%, 13/58), and 338 (12.1%, 7/58). Single nucleotide polymorphisms (SNP) were identified at 11 locations in the PVL genes. SNP (nucleotide 1396, A→G) and SNP (nucleotide 1546, A→G) were observed in >10 sequences. Four additional SNP were non-synonymous. Both SNP (nucleotide 16, C→A) and SNP (nucleotide 62, C→T) were present in the same ST59 strain. SNP (nucleotide 527, A→G) was present in five strains belonging to ST30, 121, 1, and 93. SNP (nucleotide 1436, A→C) was present in one ST30 strain. Fifteen strains belonging to ST910, ST217, and ST30 carried a PVL phage that had an icosahedral head morphology. Nine ST59 strains carried ϕ108PVL. Three ST88 strains carried a PVL phage that had an elongated head morphology. Twenty-seven strains, including 60.9% (14/23) of ST59 and all ST338 strains, had no detectable phage. In conclusion, sequence variation in PVL genes and PVL-encoding phages was generally related to the lineage. ST59 strains may indeed carry novel PVL phages., (© 2012 The Societies and Blackwell Publishing Asia Pty Ltd.)

Methicillin-resistant strains of Staphylococcus aureus (MRSA) are now the most commonly reported antibiotic-resistant bacterium in clinical settings. Therefore, there is an urgent need to develop novel antibacterial agents to control this pathogen. Bacteriophage therapy is a potential alternative treatment for MRSA infections. The objective of this study was characterization of a novel virulent bacteriophage (MSA6) isolated from a cow with mastitis. Electron microscopy showed its resemblance to members of the family Myoviridae, with an isometric head (66 nm) and a long contractile tail (173 nm). The genome of phage MSA6 was tested by pulsed-field gel electrophoresis and estimated to be about 143 kb. It exhibited rapid adsorption (>82% in 5 min), a short latent period (15 min) and a relatively small burst size (23 PFU/cell). Isolated phage was capable of infecting a wide spectrum of staphylococcal strains of both human and bovine origin. The results of this investigation indicate that MSA6 is similar to other bacteriophages belonging to the family Myoviridae (Twort, K, G1, 812) that have been successfully used in bacteriophage therapy.

We studied the usefulness of the restriction-modification (RM) test, staphylococcal cassette chromosome (SCC) mec types, and Panton-Valentine leukocidin (PVL)-encoding phages to identify Staphylococcus aureus methicillin-resistant lineages and to differentiate clones that belong to the same lineage. A total of 108 methicillin-resistant S. aureus (MRSA) strains were characterized by pulsed-field gel electrophoresis (PFGE)--multi-locus sequence typing (MLST)--spa-typing. The RM correctly identified the lineages CC5, CC8, CC30, and CC398, but not CC25 and CC72. The SCCmec and RM combined analysis allowed differentiation between MLST types within the same lineage. Only 5 MRSA strains were PVL-positive. Four PVL-positive USA300 isolates carried elongated-head type PVL-encoding phages, while the sequence type (ST)-30 strain carried an icosahedral-head phage. The combination of the RM test method, SCCmec types, and PVL phage identification could be useful for MRSA typing purposes.

Lytic bacteriophages (phages) have been investigated as treatments for bacterial infectious diseases. An induced phage, SAP-26, was isolated from a clinical isolate of Staphylococcus aureus. It belongs to the family Siphoviridae and its genome consists of double-stranded 41,207 bp DNA coding for 63 open reading frames. The phage SAP-26 showed a wide spectrum of lytic activity against both methicillin-resistant S. aureus and methicillin-susceptible S.aureus. Furthermore, combined treatment with a phage and antimicrobial agents showed a strong biofilm removal effect which induced structural changes in the biofilm matrix and a substantial decrease in the number of bacteria. Such a broad host range in S. aureus and biofilm removal activity of the phage SAP-26 suggests the possibility of its use as a therapeutic phage in combination with appropriate antimicrobial agent(s). Among the three antimicrobial agents combined with phage, the combination of rifampicin showed the best biofilm removal effect. To the authors' knowledge, this study showed for the first time that S. aureus biofilm could be efficiently eradicated with the mixture of phage and an antimicrobial agent, especially rifampicin.

The genes lukS-PV and lukF-PV for Panton-Valentine leukocidin (PVL) that confers high virulence to Staphylococcus aureus are located on the prophages (PVL phages) which have been classified into group 1 and 2 sfi21-like Siphoviridae. We report novel PVL phages lysogenized in ST59 methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in Japan (JCSC7247) and Taiwan (JCSC5967). The genomes of φ7247PVL and φ5967PVL showed more than 99% identity, and the regions containing the five genes located at both ends of the prophages, int (integrase), hol (holin), ami (amidase), lukS-PV, and lukF-PV, are highly homologous to extant PVL phages. The genes for the structural module are less homologous to these phages, but are highly homologous to non-PVL phages belonging to group 3 Sfi21-like Siphoviridae, for example φN315. Subsequent PCR identification and nucleotide sequencing of an additional 11 Taiwanese ST59 MRSA isolates suggested they all carry the same phage as φ5967PVL, which differed from φ7247PVL by a single base. This study adds evidence to the notion that novel PVL phages would be generated through illegitimate recombination events by acquiring the region at which hol, ami, luk, and int genes would line up upon lytic growth, and suggests that the PVL-positive MRSA clones that have emerged worldwide may carry distinct phages., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Staphylococcus aureus causes a wide range of suppurative infections in humans and animals. Due to its high virulence, ability to adopt various environmental conditions, and acquired multiple drug resistance, treatment of such infections has become difficult. Therefore, there is an immense need to develop alternate drug modalities to control this pathogen. In past few years, phage-encoded endolysin therapy has emerged as a new hope not only due to its ability to specifically kill the target bacteria irrespective of their antibiotic sensitivity but also because of minimum or no side effects, a problem associated with antibiotic therapy. In this article, we report purification of a broad spectrum anti-staphylococcal endolysin (P-27/HP endolysin) encoded by phage P-27/HP isolated from sewage water. On SDS-PAGE endolysin resolved in three polypeptides of molecular weights 33.5, 48.6, and 62.2 kDa. Endolysin exhibited maximum in vitro lytic activity at temperature between 35 and 40°C and pH 7.0. In vivo experiments revealed considerable (99.9%) elimination of S. aureus 27/HP from spleens of endolysin-treated mice and had saved them from death due to bacteremia caused by S. aureus 27/HP challenge infection. Thus, P-27/HP endolysin offers suitable substitute of antibiotics to control S. aureus infections.

Bacteriophage TEM126, a newly isolated temperate phage from a mitomycin-C-induced lysate of wild-type Staphylococcus aureus isolated from food, has an isometric head, a noncontractile tail, and a double-stranded DNA genome with a length of 33,540 bp and a G+C content of 33.94%. Bioinformatics analysis of the phage genome revealed 44 putative open reading frames (ORFs). Predicted protein products of the ORFs were determined and described. Temperate phage TEM126 can be classified as a member of the family Siphoviridae by morphology and genome structure. Temperate phage TEM126 showed 84% similarity with Staphylococcus phage phiNM1. To our knowledge, this is the first report of genomic sequencing and characterization of temperate phage TEM126 from a wild-type S. aureus isolated from foods in Korea.

Genetically diverse community-associated methicillin resistant Staphylococcus aureus (CA-MRSA) can harbor a bacteriophage encoding Panton-Valentine leukocidin (PVL) lysogenized into its chromosome (prophage). Six PVL phages (ΦPVL, Φ108PVL, ΦSLT, ΦSa2MW, ΦSa2USA, and ΦSa2958) are known, and single-nucleotide polymorphisms (SNPs) in the PVL genes have been reported. We sought to determine the distribution of lysogenized PVL phages among MRSA strains with PVL (PVL-MRSA strains), the PVL gene sequences, and the chromosomal phage insertion sites in 114 isolates comprising nine clones of PVL-MRSA that were selected for maximal underlying genetic diversity. The six PVL phages were identified by PCR; ΦSa2USA was present in the highest number of different lineages (multilocus sequence type clonal complex 1 [CC1], CC5, CC8, and sequence type 93 [ST93]) (n = 37 isolates). Analysis of 92 isolates confirmed that PVL phages inserted into the same chromosomal insertion locus in CC22, -30, and -80 but in a different locus in isolates of CC1, -5, -8, -59, and -88 and ST93 (and CC22 in two isolates). Within the two different loci, specific attachment motifs were found in all cases, although some limited inter- and intralineage sequence variation occurred. Overall, lineage-specific relationships between the PVL phage, the genes that encode the toxin, and the position at which the phage inserts into the host chromosome were identified. These analyses provide important insights into the microepidemiology of PVL-MRSA, will prove a valuable adjunct in outbreak investigation, and may help predict the emergence of new strains.

Emergence of multidrug resistant strains has created serious problem for safe eradication of Staphylococcus aureus infections. Therefore, there is an urgent need to develop novel antibacterial agents to control this pathogen. Bacteriophages kill bacteria irrespective of their antibiotic sensitivity and thus they can be used as potent prophylactic/therapeutic agent to treat such infections. Here, we report isolation of broad host range anti-staphylococcal lytic bacteriophage P-27/HP from sewage water. This phage was able to inhibit 17 of 28 (60%) human disease associated S. aureus isolates. In vitro studies revealed its strong lytic efficacy to diminish S. aureus 27/HP population (c.f.u.) by more than 5.0 logs (P < 0.0001) (equivalent to 99.99%) in 3 h at 0.01 MOI. In vivo lytic efficacy analysis showed that a single subcutaneous injection of phage P-27/HP (10(7) p.f.u.) was sufficient to protect S. aureus 27/HP infected (5 × 10(8) c.f.u.) mice from bacteremia and subsequent death. A considerable decline of more than 6 logs (99.9%) in splenic S. aureus 27/HP c.f.u. count was noted at the 3 days of phage treatment. In conclusion, our results suggest that phage P-27/HP is polyvalent in nature and has high-lytic potential towards S. aureus, thus, a therapy employing this phage would be efficacious to control S. aureus infections.

Phage-coded lysin is an enzyme that destroys the cell walls of bacteria. Phage lysin could be an alternative to conventional antibiotic therapy against pathogens that are resistant to multiple antibiotics. In this study, a novel staphylococcal phage, GH15, was isolated, and the endogenous lytic enzyme (LysGH15) was expressed and purified. The lysin LysGH15 displayed a broad lytic spectrum; in vitro treatment killed a number of Staphylococcus aureus strains rapidly and completely, including methicillin-resistant S. aureus (MRSA). In animal experiments, a single intraperitoneal injection of LysGH15 (50 μg) administered 1 h after MRSA injections at double the minimum lethal dose was sufficient to protect mice (P < 0.01). Bacteremia in unprotected mice reached colony counts of about 10(7) CFU/ml within 3.5 h after challenge, whereas the mean colony count in lysin-protected mice was less than 10(4) CFU/ml (and ultimately became undetectable). These results indicate that LysGH15 can kill S. aureus in vitro and can protect mice efficiently from bacteremia in vivo. The phage lysin LysGH15 might be an alternative treatment strategy for infections caused by MRSA.