Bacteriophage therapy for drug-resistant Staphylococcus aureus infections.

Drug-resistant Staphylococcus aureus stands as a prominent pathogen in nosocomial and community-acquired infections, capable of inciting various infections at different sites in patients. This includes Staphylococcus aureus bacteremia (SaB), which exhibits a severe infection frequently associated with significant mortality rate of approximately 25%. In the absence of better alternative therapies, antibiotics is still the main approach for treating infections. However, excessive use of antibiotics has, in turn, led to an increase in antimicrobial resistance. Hence, it is imperative that new strategies are developed to control drug-resistant S. aureus infections. Bacteriophages are viruses with the ability to infect bacteria. Bacteriophages, were used to treat bacterial infections before the advent of antibiotics, but were subsequently replaced by antibiotics due to limited theoretical understanding and inefficient preparation processes at the time. Recently, phages have attracted the attention of many researchers again because of the serious problem of antibiotic resistance. This article provides a comprehensive overview of phage biology, animal models, diverse clinical case treatments, and clinical trials in the context of drug-resistant S. aureus phage therapy. It also assesses the strengths and limitations of phage therapy and outlines the future prospects and research directions. This review is expected to offer valuable insights for researchers engaged in phage-based treatments for drug-resistant S. aureus infections.

Targeted Antimicrobial Photodynamic Therapy of Biofilm-Embedded and Intracellular Staphylococci with a Phage Endolysin's Cell Binding Domain.

Bacterial pathogens are progressively adapting to current antimicrobial therapies with severe consequences for patients and global health care systems. This is critically underscored by the rise of methicillin resistant Staphylococcus aureus (MRSA) and other biofilm-forming staphylococci. Accordingly, alternative strategies have been explored to fight such highly multidrug resistant microorganisms, including antimicrobial photodynamic therapy (aPDT) and phage therapy. aPDT has the great advantage that it does not elicit resistance, while phage therapy allows targeting of specific pathogens. In the present study, we aimed to merge these benefits by conjugating the cell-binding domain (CBD3) of a Staphylococcus aureus phage endolysin to a photoactivatable silicon phthalocyanine (IRDye 700DX) for the development of a Staphylococcus-targeted aPDT approach. We show that, upon red-light activation, the resulting CBD3-700DX conjugate generates reactive oxygen species that effectively kill high loads of planktonic and biofilm-resident staphylococci, including MRSA. Furthermore, CBD3-700DX is readily internalized by mammalian cells, where it allows the targeted killing of intracellular MRSA upon photoactivation. Intriguingly, aPDT with CBD3-700DX also affects mammalian cells with internalized MRSA, but it has no detectable side effects on uninfected cells. Altogether, we conclude that CBD3 represents an attractive targeting agent for Staphylococcus-specific aPDT, irrespective of planktonic, biofilm-embedded, or intracellular states of the bacterium. IMPORTANCE Antimicrobial resistance is among the biggest threats to mankind today. There are two alternative antimicrobial therapies that may help to control multidrug-resistant bacteria. In phage therapy, natural antagonists of bacteria, lytic phages, are harnessed to fight pathogens. In antimicrobial photodynamic therapy (aPDT), a photosensitizer, molecular oxygen, and light are used to produce reactive oxygen species (ROS) that inflict lethal damage on pathogens. Since aPDT destroys multiple essential components in targeted pathogens, aPDT resistance is unlikely. However, the challenge in aPDT is to maximize target specificity and minimize collateral oxidative damage to host cells. We now present an antimicrobial approach that combines the best features of both alternative therapies, namely, the high target specificity of phages and the efficacy of aPDT. This is achieved by conjugating the specific cell-binding domain from a phage protein to a near-infrared photosensitizer. aPDT with the resulting conjugate shows high target specificity toward MRSA with minimal side effects.

Bacteriophage Therapy for the Prevention and Treatment of Fracture-Related Infection Caused by Staphylococcus aureus: a Preclinical Study.

Although several studies have shown promising clinical outcomes of phage therapy in patients with orthopedic device-related infections, questions remain regarding the optimal application protocol, systemic effects, and the impact of the immune response. This study provides a proof-of-concept of phage therapy in a clinically relevant rabbit model of fracture-related infection (FRI) caused by Staphylococcus aureus. In a prevention setting, phage in saline (without any biomaterial-based carrier) was highly effective in the prevention of FRI, compared to systemic antibiotic prophylaxis alone. In the subsequent study involving treatment of established infection, daily administration of phage in saline through a subcutaneous access tube was compared to a single intraoperative application of a phage-loaded hydrogel and a control group receiving antibiotics only. In this setting, although a possible trend of bacterial load reduction on the implant was observed with the phage-loaded hydrogel, no superior effect of phage therapy was found compared to antibiotic treatment alone. The application of phage in saline through a subcutaneous access tube was, however, complicated by superinfection and the development of neutralizing antibodies. The latter was not found in the animals that received the phage-loaded hydrogel, which may indicate that encapsulation of phages into a carrier such as a hydrogel limits their exposure to the adaptive immune system. These studies show phage therapy can be useful in targeting orthopedic device-related infection, however, further research and improvements of these application methods are required for this complex clinical setting. IMPORTANCE Because of the growing spread of antimicrobial resistance, the use of alternative prevention and treatment strategies is gaining interest. Although the therapeutic potential of bacteriophages has been demonstrated in a number of case reports and series over the past decade, many unanswered questions remain regarding the optimal application protocol. Furthermore, a major concern during phage therapy is the induction of phage neutralizing antibodies. This study aimed at providing a proof-of-concept of phage therapy in a clinically relevant rabbit model of fracture-related infection caused by Staphylococcus aureus. Phage therapy was applied as prophylaxis in a first phase, and as treatment of an established infection in a second phase. The development of phage neutralizing antibodies was evaluated in the treatment study. This study demonstrates that phage therapy can be useful in targeting orthopedic device-related infection, especially as prophylaxis; however, further research and improvements of these application methods are required.

A potent enzybiotic against methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus is one of the most dreadful infectious agents, responsible for high mortality and morbidity in both humans and animals. The increased prevalence of multidrug-resistant (MDR) Staphylococcus aureus strains has limited the number of available treatment options, which calls for the development of alternative and effective modalities against MDR S. aureus. Endolysins are bacteriophage-derived antibacterials, which attack essential conserved elements of peptidoglycan that are vital for bacterial survival, making them promising alternatives or complements to existing antibiotics for tackling such infections. For developing endolysin lysin-methicillin-resistant-5 (LysMR-5) as an effective antimicrobial agent, we evaluated its physical and chemical characteristics, and its intrinsic antibacterial activity against staphylococcal strains, including methicillin-resistant Staphylococcus aureus (MRSA). In this study, we cloned, expressed, and purified LysMR-5 from S. aureus phage MR-5. In silico analysis revealed that LysMR-5 harbors two catalytic and one cell wall-binding domain. Biochemical characterization and LC-MS analysis showed that both catalytic domains were active and had no dependence on divalent ions for their action, Zn2+ exerted a negative effect. The optimal lytic activity of the endolysin was at 37 °C/pH 7.0 and in the presence of ≥ 300 mM concentration of NaCl. Circular dichroism (CD) demonstrated a loss in secondary structure with an increase in temperature confirming the thermosensitive nature of endolysin. Antibacterial assays revealed that LysMR-5 was active against diverse clinical isolates of staphylococci. It showed high lytic efficacy against S. aureus ATCC 43300, as an endolysin concentration as low as 15 µg/ml was sufficient to achieve maximum lytic activity within 30 min and it was further confirmed by scanning electron microscopy. Our results indicate that rapid and strong bactericidal activity of LysMR-5 makes it a valuable candidate for eradicating multidrug-resistant S. aureus.

Antimicrobial effect of commercial phage preparation Stafal® on biofilm and planktonic forms of methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus may be a highly virulent human pathogen, especially when it is able to form a biofilm, and it is resistant to antibiotic. Infections caused by these bacteria significantly affect morbidity and mortality, primarily in hospitalized patients. Treatment becomes more expensive, more toxic, and prolonged. This is the reason why research on alternative therapies should be one of the main priorities of medicine and biotechnology. A promising alternative treatment approach is bacteriophage therapy. The effect of the anti-staphylococcal bacteriophage preparation Stafal® on biofilm reduction was assessed on nine S. aureus strains using both sonication with subsequent quantification of surviving cells on the catheter surface and evaluation of biofilm reduction in microtiter plates. It was demonstrated that the bacteriophages destroy planktonic cells very effectively. However, to destroy cells embedded in the biofilm effectively requires a concentration at least ten times higher than that provided by the commercial preparation. The catheter disc method (CDM) allowed easier comparison of the effect on planktonic cells and cells in a biofilm than the microtiter plate (MTP) method.

Isolation and characterization of a potentially novel Siphoviridae phage (vB_SsapS-104) with lytic activity against Staphylococcus saprophyticus isolated from urinary tract infection.

Antibiotic resistance is increasing among Staphylococcus saprophyticus strains isolated from urinary tract infection. This necessitates alternative therapies. For this, a lytic phage (vB_SsapS-104) against S. saprophyticus, which formed round and clear plaques on bacterial culture plates, was isolated from hospital wastewater and characterized. Microscopy analysis showed that it had a small head (about 50 nm), tail (about 80 nm), and a collar (about 22 nm in length and 19 nm in width) indicating to be a phage within Siphoviridae family. Phage vB_SsapS-104 showed a large latency period of about 40 min, rapid adsorption rate that was significantly enhanced by MgCl2 and CaCl2, and high stability to a wide range of temperatures and pH values. Restriction analyses demonstrated that phage consists of a double-stranded DNA with an approximate genome size of 40 Kb. BLAST results did not show high similarity (megablast) with other previously identified phages. But, in Blastn, similarity with Staphylococcus phages was observed. Phage vB_SsapS-104 represented high anti-bacterial activity against S. saprophyticus isolates in vitro as it was able to lyse 8 of the 9 clinical isolates (%88.8) obtained from a hospital in Gorgan, Iran. It was a S. saprophyticus-specific phage because no lytic activity was observed on some other pathogenic bacteria tested. Therefore, phage vB_SsapS-104 can be considered as a specific virulent phage against of S. saprophyitcus isolated from urinary tract infection. This study provided the partial genomic characterization of S. saprophyticus phage and its application against urinary tract infection associated with S. saprophyticus. This phage also can be considered as a good candidate for a therapeutic alternative in the future.

Are Phage Lytic Proteins the Secret Weapon To Kill Staphylococcus aureus?

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most threatening microorganisms for global human health. The current strategies to reduce the impact of S. aureus include a restrictive control of worldwide antibiotic use, prophylactic measures to hinder contamination, and the search for novel antimicrobials to treat human and animal infections caused by this bacterium. The last strategy is currently the focus of considerable research. In this regard, phage lytic proteins (endolysins and virion-associated peptidoglycan hydrolases [VAPGHs]) have been proposed as suitable candidates. Indeed, these proteins display narrow-spectrum antimicrobial activity and a virtual lack of bacterial-resistance development. Additionally, the therapeutic use of phage lytic proteins in S. aureus animal infection models is yielding promising results, showing good efficacy without apparent side effects. Nonetheless, human clinical trials are still in progress, and data are not available yet. This minireview also analyzes the main obstacles for introducing phage lytic proteins as human therapeutics against S. aureus infections. Besides the common technological problems derived from large-scale production of therapeutic proteins, a major setback is the lack of a proper legal framework regulating their use. In that sense, the relevant health authorities should urgently have a timely discussion about these new antimicrobials. On the other hand, the research community should provide data to dispel any doubts regarding their efficacy and safety. Overall, the appropriate scientific data and regulatory framework will encourage pharmaceutical companies to invest in these promising antimicrobials.

Efficacy of lytic Staphylococcus aureus bacteriophage against multidrug-resistant Staphylococcus aureus in mice.

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.

Combination Therapy of LysGH15 and Apigenin as a New Strategy for Treating Pneumonia Caused by Staphylococcus aureus.

Pneumonia is one of the most prevalent Staphylococcus aureus-mediated diseases, and the treatment of this infection is becoming challenging due to the emergence of multidrug-resistant S. aureus, especially methicillin-resistant S. aureus (MRSA) strains. It has been reported that LysGH15, the lysin derived from phage GH15, displays high efficiency and a broad lytic spectrum against MRSA and that apigenin can markedly diminish the alpha-hemolysin of S. aureus. In this study, the combination therapy of LysGH15 and apigenin was evaluated in vitro and in a mouse S. aureus pneumonia model. No mutual adverse influence was detected between LysGH15 and apigenin in vitro. In animal experiments, the combination therapy showed a more effective treatment effect than LysGH15 or apigenin monotherapy (P < 0.05). The bacterial load in the lungs of mice administered the combination therapy was 1.5 log units within 24 h after challenge, whereas the loads in unprotected mice or mice treated with apigenin or LysGH15 alone were 10.2, 4.7, and 2.6 log units, respectively. The combination therapy group showed the best health status, the lowest ratio of wet tissue to dry tissue of the lungs, the smallest amount of total protein and cells in the lung, the fewest pathological manifestations, and the lowest cytokine level compared with the other groups (P < 0.05). With regard to its better protective efficacy, the combination therapy of LysGH15 and apigenin exhibits therapeutic potential for treating pneumonia caused by MRSA. This paper reports the combination therapy of lysin and natural products derived from traditional Chinese medicine.

Antibacterial activity of the alkaloid extract and isolated compounds from Croton bonplandianum Baill. (Euphorbiaceae) / Atividade anti-bacteriana do extrato alcaloide e de compostos isolados de Croton bonplandianum Baill. (Euphorbiaceae)

ABSTRACT The antibacterial activity of the alkaloid extract from the leaves of Croton bonplandianum Baill. and its main compounds, sparsiflorine and crotsparine, was tested against Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa by the resazurin microtitre-plate method. Pure compounds were identified by spectroscopic techniques, mainly 1D and 2D NMR. The alkaloid extract showed activity particularly against the S. aureus and P. aeruginosa. Regarding the pure compounds, the crotsparine was inactive against the microorganisms assayed, whereas the sparsiflorine indicated a moderate activity similar to the alkaloid extract. The Pseudomonas aeruginosa was the most sensitive of the tested microorganisms with MIC of 0.141 mg/mL. The results suggest that the activity of the extract may be credited mainly to the presence of the sparsiflorine. Although the activity of the sparsiflorine does not get close to the antimicrobial drugs in clinical use, it still could be a lead compound for the development of new antibacterial substances.

RESUMO A atividade antibacteriana do extrato alcaloidal das folhas de Croton bonplandianum Baill., além dos principais compostos isolados, esparsiflorina e crotsparina, foi testada contra Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae e Pseudomonas aeruginosa. Foi utilizado o método de microdiluição em placa empregando resazurina como indicador da viabilidade celular. Os compostos isolados foram identificados por técnicas espectroscópicas, principalmente RMN 1D e 2D. O extrato alcaloidal foi ativo principalmente contra S. aureus e P. aeruginosa. Crotsparina mostrou-se inativa contra todos os micro-organismos testados, enquanto esparsiflorina apresentou atividade moderada, a qual foi similar à do extrato bruto. Pseudomonas aeruginosa foi a mais sensível das bactérias testadas, com CIM de 0,141 mg/mL. Os resultados sugerem que a atividade do extrato pode ser devida em grande medida pela presença de esparsiflorina. Apesar de a CIM da esparsiflorina não ter se aproximado daquela apresentada pelos agentes antimicrobianos em uso clínico, tal composto ainda pode compor um protótipo interessante para o desenvolvimento de novas substâncias antibacterianas.

Bacteriophage as effective decolonising agent for elimination of MRSA from anterior nares of BALB/c mice.

BACKGROUND: Nasal carriers not only pose serious threat to themselves but also to the community by playing an active role in the dissemination of serious and life threatening S. aureus especially MRSA strains. The present study focuses on the use of broad spectrum lytic phage as decolonising agent. In addition, the combined use of lytic phage with mupirocin has also been investigated as an effective decolonising regimen. The effect of phage on the adherence, invasion and cytotoxic effect of MRSA strains on nasal epithelial cells was studied in an ex-vivo model of cultured murine nasal epithelial cells. This was followed by demonstration of therapeutic potential of phage along with mupirocin in decolonising the nares of BALB/c mice using a nasal model of MRSA colonisation. RESULTS: Phage was able to significantly reduce the in vitro adherence, invasion and cytotoxicity of MRSA 43300 as well as other clinical MRSA strains on murine nasal epithelial cells as compared to untreated control. Also, the frequency of emergence of spontaneous mutants decreased to negligible levels when both the agents (phage and mupirocin) were used together. CONCLUSION: Phage MR-10, given along with mupirocin showed an additive effect and the combination was able to effectively eradicate the colonising MRSA population from the nares of mice by day 5.

Facing antibiotic resistance: Staphylococcus aureus phages as a medical tool.

Staphylococcus aureus is a common and often virulent pathogen in humans. This bacterium is widespread, being present on the skin and in the nose of healthy people. Staphylococcus aureus can cause infections with severe outcomes ranging from pustules to sepsis and death. The introduction of antibiotics led to a general belief that the problem of bacterial infections would be solved. Nonetheless, pathogens including staphylococci have evolved mechanisms of drug resistance. Among current attempts to address this problem, phage therapy offers a promising alternative to combat staphylococcal infections. Here, we present an overview of current knowledge on staphylococcal infections and bacteriophages able to kill Staphylococcus, including experimental studies and available data on their clinical use.

Bacteriophage reduces biofilm of Staphylococcus aureus ex vivo isolates from chronic rhinosinusitis patients.

BACKGROUND: Staphylococcus aureus is the most common organism in recalcitrant chronic rhinosinusitis (CRS) and is often resistant to traditional antibiotic therapy. Bacteriophages ("phages") are a potential candidate for a new, effective therapy. For phages to be useful in the setting of CRS, two minimum requirements must be presented: (1) phages must be effective against S. aureus biofilms and (2) phages must have a broad spectrum of activity. This study aimed to assess the in vitro activity of a phage cocktail (CockTail of Staphylococcus aureus specific bacteriophage [CT-SA]) against S. aureus biofilms and a broad panel of strains isolated from patients with CRS. METHODS: The study examined 66 clinical isolates (CIs) of S. aureus. All isolates were tested for the susceptibility to phage lysis by spotting CT-SA onto bacterial lawns. To measure its effect on S. aureus biofilms, a minimum biofilm eradication concentration assay was used, using five S. aureus isolates. Biofilms of these isolates were grown, treated with CT-SA for 48 hours, fluorescently stained, and viewed using confocal scanning laser microscopy. RESULTS: CT-SA lysed 62 of 66 (94%) CIs of S. aureus. CT-SA treatment yielded significant reductions in biofilm mass for 4/5 CIs tested and for ATCC 25923. Challenge of S. aureus with a single phage resulted in the emergence of bacteriophage-insensitive mutants (BIM) with a frequency of 10(-7), and challenge with CT-SA completely prevented their development. CONCLUSION: This study indicates that phage cocktail CT-SA can effectively eliminate S. aureus, in planktonic and biofilm forms, from the great majority of CIs from this hospital setting. In addition, its potential effect in preventing the emergence of BIMs was also established. Thus, CT-SA has the potential to treat S. aureus infection and biofilm in CRS patients.

Experimental phage therapy against lethal lung-derived septicemia caused by Staphylococcus aureus in mice.

Nosocomial respiratory infections caused by methicillin-resistant Staphylococcus aureus (MRSA) can progress to lethal systemic infections. Bacteriophage (phage) therapy is expected to be effective against these critical infections. Previously, phage S13' was proposed as a potential therapeutic phage. We here examined phage treatment in a mouse model of lung-derived septicemia using phage S13'. Intraperitoneal phage administration at 6 h postinfection reduced the severity of infection and rescued the infected mice. Phage S13' can efficiently lyse hospital-acquired MRSA strains causing pneumonia-associated bacteremia in vitro. Thus, phage therapy may be a possible therapeutic intervention in staphylococcal lung-derived septicemia.

Safety and efficacy of topical bacteriophage and ethylenediaminetetraacetic acid treatment of Staphylococcus aureus infection in a sheep model of sinusitis.

BACKGROUND: Treatment of sinonasal bacterial biofilms continues to be a challenge in modern rhinology. This study's objective was to assess the safety and efficacy of topically applied Cocktail of S. aureus specific phage (CTSA) alone and in combination with ethylenediaminetetraacetic acid (EDTA) for treatment of Staphylococcus aureus biofilms in vivo. METHODS: Using a sheep model of sinusitis, frontal sinuses (n = 6 per treatment) were flushed once daily with a CTSA (2 × 10(6) plaque forming units [PFU]/mL), with or without EDTA (0.075 mg/mL), and compared to a control flush containing saline and heat-inactivated CTSA. Safety was assessed using histology and scanning electron microscopy (SEM) after treatment for 3 days. Efficacy was assessed by quantifying the generation of S. aureus biofilms in the frontal sinuses after 5 days of treatment. Biofilm mass was compared between treatment groups and controls using LIVE/DEAD BacLight staining and confocal scanning laser microscopy to visualize the tissue sections. COMSTAT2 software was used to compute the biofilm mass present on tissue sections. RESULTS: Tissue morphology was conserved, with no significant signs of inflammation, when comparing control and test treatments. Furthermore, SEM analysis indicated test treatments were not toxic or damaging to mucosal cilia. COMSTAT2 quantification of biofilm showed a significant reduction in biofilm levels when comparing the control with CTSA (p = 0.0043), EDTA (p = 0.0095), and CTSA-EDTA (p = 0.0022) treatments. CONCLUSION: Results indicate that CTSA and EDTA are safe and efficacious for short-term topical application against S. aureus infection in a sheep sinusitis model, and have the potential to be translated to a clinical setting.

Bacteriophage therapy against staphylococci.

The emergence of the multi-drug-resistant Staphylococcus aureus strains has prompted interest in alternatives to conventional drugs. Among the possible options one of the most promising is the therapeutic use of bacteriophages. Over the recent decades, increasing amount of literature has validated the use of bacteriophages for therapy and prophylaxis against drug-resistant staphylococci. This work attempts to review the current knowledge on bacteriophages and their usages for treatment of staphylococcal diseases.

Romulus and Remus, two phage isolates representing a distinct clade within the Twortlikevirus genus, display suitable properties for phage therapy applications.

The renewed interest in controlling Staphylococcus aureus infections using their natural enemies, bacteriophages, has led to the isolation of a limited number of virulent phages so far. These phages are all members of the Twortlikevirus, displaying little variance. We present two novel closely related (95.9% DNA homology) lytic myoviruses, Romulus and Remus, with double-stranded DNA (dsDNA) genomes of 131,333 bp and 134,643 bp, respectively. Despite their relatedness to Staphylococcus phages K, G1, ISP, and Twort and Listeria phages A511 and P100, Romulus and Remus can be proposed as isolates of a new species within the Twortlikevirus genus. A distinguishing feature for these phage genomes is the unique distribution of group I introns compared to that in other staphylococcal myoviruses. In addition, a hedgehog/intein domain was found within their DNA polymerase genes, and an insertion sequence-encoded transposase exhibits splicing behavior and produces a functional portal protein. From a phage therapy application perspective, Romulus and Remus infected approximately 70% of the tested S. aureus isolates and displayed promising lytic activity against these isolates. Furthermore, both phages showed a rapid initial adsorption and demonstrated biofilm-degrading capacity in a proof-of-concept experiment.

Genomics of staphylococcal Twort-like phages--potential therapeutics of the post-antibiotic era.

Polyvalent bacteriophages of the genus Twort-like that infect clinically relevant Staphylococcus strains may be among the most promising phages with potential therapeutic applications. They are obligatorily lytic, infect the majority of Staphylococcus strains in clinical strain collections, propagate efficiently and do not transfer foreign DNA by transduction. Comparative genomic analysis of 11 S. aureus/S. epidermidis Twort-like phages, as presented in this chapter, emphasizes their strikingly high similarity and clear divergence from phage Twort of the same genus, which might have evolved in hosts of a different species group. Genetically, these phages form a relatively isolated group, which minimizes the risk of acquiring potentially harmful genes. The order of genes in core parts of their 127 to 140-kb genomes is conserved and resembles that found in related representatives of the Spounavirinae subfamily of myoviruses. Functions of certain conserved genes can be predicted based on their homology to prototypical genes of model spounavirus SPO1. Deletions in the genomes of certain phages mark genes that are dispensable for phage development. Nearly half of the genes of these phages have no known homologues. Unique genes are mostly located near termini of the virion DNA molecule and are expressed early in phage development as implied by analysis of their potential transcriptional signals. Thus, many of them are likely to play a role in host takeover. Single genes encode homologues of bacterial virulence-associated proteins. They were apparently acquired by a common ancestor of these phages by horizontal gene transfer but presumably evolved towards gaining functions that increase phage infectivity for bacteria or facilitate mature phage release. Major differences between the genomes of S. aureus/S. epidermidis Twort-like phages consist of single nucleotide polymorphisms and insertions/deletions of short stretches of nucleotides, single genes, or introns of group I. Although the number and location of introns may vary between particular phages, intron shuffling is unlikely to be a major factor responsible for specificity differences.

Characterization of a bacteriophage, isolated from a cow with mastitis, that is lytic against Staphylococcus aureus strains.

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.