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.

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.

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.

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.

Potential of bacteriophages and their lysins in the treatment of MRSA: current status and future perspectives.

Bacteriophages (phages) are viruses that specifically infect and kill bacteria. Lysins are enzymes of bacteriophage origin that cleave covalent bonds in peptidoglycan, thereby inducing rapid lysis of a bacterial cell. As potential antibacterial agents, phages and lysins have some important features in common, especially the capacity to kill antibiotic-resistant bacteria, a narrow antibacterial range, and lack of toxic effects on mammalian cells. In this article we present the staphylococcal phages and their lysins that can be used to combat methicillin-resistant Staphylococcus aureus (MRSA), one of today's most dangerous pathogens. We also discuss the use of phages as vectors specifically delivering different antibacterial agents to bacterial cells. Experimental data show that both phages and lysins could be effective in the treatment of MRSA.

Bacteriophage therapy for Staphylococcus aureus bacteremia in streptozotocin-induced diabetic mice.

The protective effect of bacteriophage was assessed against experimental Staphylococcus aureus lethal bacteremia in streptozotocin (STZ) induced-diabetic and non-diabetic mice. Intraperitoneal administrations of S. aureus (RCS21) of 2 × 108 CFU caused lethal bacteremia in both diabetic and non-diabetic mice. A single administration of a newly isolated lytic phage strain (GRCS) significantly protected diabetic and non-diabetic mice from lethal bacteremia (survival rate 90% and 100% for diabetic and non-diabetic bacteremic groups versus 0% for saline-treated groups). Comparison of phage therapy to oxacillin treatment showed a significant decrease in RCS21 of 5 and 3 log units in diabetic and non-diabetic bacteremic mice, respectively. The same protection efficiency of phage GRCS was attained even when the treatment was delayed up to 4 h in both diabetic and non-diabetic bacteremic mice. Inoculation of mice with a high dose (10¹° PFU) of phage GRCS alone produced no adverse effects attributable to the phage per se. These results suggest that phages could constitute valuable prophylaxis against S. aureus infections, especially in immunocompromised patients.

Phage therapy of staphylococcal infections (including MRSA) may be less expensive than antibiotic treatment.

The current drama of antibiotic resistance has revived interest in phage therapy. In response to this challenge, a phage therapy center was established at our Institute in 2005 which accepts patients from Poland and abroad with antibiotic-resistant infections. We now present data showing that efficient phage therapy of staphylococcal infections is no longer a treatment of last resort (when all antibiotics fail), but allows for significant savings in the costs of healthcare.

The specific and sensitive detection of bacterial pathogens within 4 h using bacteriophage amplification.

This paper describes a novel approach, termed the 'phage amplification assay', for the rapid detection and identification of specific bacteria. The technique is based on the phage lytic cycle with plaque formation as the assay end-point. It is highly sensitive, quantitative and gives results typically within 4 h. The assay comprises four main stages: (1) phage infection of target bacterium; (2) destruction of exogenous phage; (3) amplification of phage within infected host and (4) plaque formation from infected host with the aid of helper bacteria. A key component of this assay is a potent virucidal agent derived from natural plant extracts, pomegranate rind extract (PRE). In combination with ferrous sulphate PRE can bring about an 11 log-cycle reduction in phage titre within 3 min. This is achieved without any injury to the infected target bacteria. Subsequently, any resulting plaques are derived only from infected target organisms. Data are presented for a range of bacterial hosts including Pseudomonas aeruginosa, Salmonella typhimurium and Staphylococcus aureus. The detection limit for Ps. aeruginosa was 40 bacteria ml-1 in a time of 4 h and 600 bacteria m-1 for Salm. typhimurium. Application of the principles of this technology to other bacterial genera is discussed.