Bacteriophage K for reduction of Staphylococcus aureusbiofilm on central venous catheter material

Abstract

The purpose of this project was to determine whether bacteriophage can reduce bacterial colonization and biofilm formation on central venous catheter material. Twenty silicone discs were inoculated for 24 h with broth culture of Methicillin sensitive staphylococcus aureus (0.5 McFarland standard). The inoculate was aspirated and discs placed into two equal groups for 24 h: (1) untreated controls; (2) bacteriophage treatment (staphylococcal bacteriophage K, propagated titer > 10⁸). At the completion of the experiment discs were processed for quantitative culture. Statistical testing was performed using the rank sum test. Mean colony forming units (CFU) were significantly decreased in experimental compared with controls (control 6.3 × 10⁵ CFU, experimental 6.7 × 10¹, P ≤ 0.0001). Application of bacteriophage to biofilm infected central venous catheter material significantly reduced bacterial colonization and biofilm presence. Our data suggests that bacteriophage treatment may be a feasible strategy for addressing central venous catheter staph aureus biofilm infections.

Introduction

Microorganisms that colonize indwelling catheters have the capability to form biofilms on the catheter surface, which are a microbially derived community of cells embedded in a matrix of extracellular polymeric substances that are irreversibly attached to a living or nonliving substratum. These biofilms form a microenvironment that confers increased antimicrobial resistance to the embedded microorganisms via a variety of mechanisms. Biofilm-associated organisms can also elicit disease processes by detachment of individual cells or aggregates of cells resulting in bloodstream infections, by production of endotoxin, or by providing a niche for the development of antibiotic-resistant organisms.^1-3

Approximately 250000 cases of intravascular catheter-related bloodstream infections occur in the United States each year, resulting in a mortality of between 12–25% with an estimated cost of treatment per episode of approximately $25000.^3-10 The standard management of catheter-related infection involves decisions regarding removal of the catheter and the administration of appropriate antibiotics. Catheter removal and eventual replacement, raises important practical problems in these patients requiring parenteral nutrition, chemotherapy, and hemodialysis, among others. To avoid catheter removal, strategies for treatment of catheter-related bacteremia with antibiotics administered through the catheter or locked within the catheter lumen have been previously studied. However, these antibiotic catheter salvage protocols are not recommended due to a high failure rate of well over 30% and the serious risk of contributing to the development of antibiotic resistant bacteria. It is widely believed that the high rate of therapy failure is explained in part by the inability of most antibiotics to kill bacteria growing in a biofilm.^3,6,9-15

Certain infections now thought to be associated with biofilms, including otitis media, urinary tract infections, periodontitis, and burn infections, have been effectively treated with phage therapy.^16-18 Phage therapy has also been proposed to be used against multidrug-resistant bacteria, and may reduce the use of antimicrobial drugs and the spread of antimicrobial resistance.^9,15,17-21 Despite the potential advantages of phage therapy, only a few studies have concentrated on its direct application toward biofilm control and treatment. Prior investigations suggest that the application of bacteriophage to indwelling medical devices, such as intravascular catheters, could provide a strategy for the reduction in biofilm formation by clinically relevant bacteria such as Staphylococcus.^19,20

The purpose of this study was to evaluate bacteriophage antimicrobial therapy application for eliminating Staphylococcus aureus biofilm on central venous catheter material.

Results

Mean colony forming units (CFU) were significantly decreased in experimental group compared with controls (control 6.3 × 10⁵ CFU, experimental 6.7 × 10¹, P ≤ 0.0001) (Fig. 1).

Figure 1. Mean CFU in the experimental group compared with controls.

Discussion

The broad objective of this research was to further investigate an alternative antimicrobial therapy for salvage treatment of long-term central venous catheter-related infection using bacteriophage. We demonstrated a significant decrease in Staphylococcus aureus biofilm formation on the surface of central venous catheter material treated with bacteriophage compared with untreated controls in an in-vitro model. Phage therapy involves the targeted application of bacteriophages that, upon encounter with specific pathogenic bacteria, can infect and kill them. In order to be successful, phage therapy must deliver sufficient phage density in the vicinity of the target bacteria in order to achieve bacterial clearance.^21,22 For this reason, concentrations of phage utilized in the current study would be needed to be clinically effective as a treatment for indwelling central venous catheter infections; a catheter “lock” technique, whereby a volume of phage solution equal to that of the catheter lumen is placed into a catheter and the then closed, sealing the solution within the indwelling catehter, has a high likelihood of delivering satisfactorily high concentrations of phage used in this experiment to the target in vivo.

Bacteriophage K, used in the current study, is a polyvalent Staphylococcus phage and capable of lysing 10 different Staphylococcus epidermidis strains and nine different Staphylococcus species including a vancomycin-resistant S. aureus (VRSA) strain and several methicillin-resistant S. aureus (MRSA) strains.^23-26 Our study utilized bacteriophage K titers in a bacteriophage lock in the lumen of colonized catheters with concentrations of greater than 10⁸ PFU/ml. Further work is needed to delineate an ideal dwell time, concentration, and systemic response to therapy, and the effectiveness of this approach in-vivo; it is virtually unknown how phage will interact when exposed to the intravenous environment in humans, and research is needed to understand what is likely to be a complex biological interaction. We also acknowledge the additional challenge of bacterial resistance to phage K, which has yet to be shown by this work, may require additional phage treatment strategies. This study is a preliminary step toward the development of a new strategy for treating central venous catheter infections, as bacteriophage therapy has never been reported, to our knowledge, for an intravascular surface.

This study has additional limitations. The small sample size may limit a broad generalization of the findings, though statistical significance was easily achieved. The biofilm in this work was artificially composed entirely of a single strain of S. aureus. However, we chose bacteriophage K as it has a broad spectrum of lytic activity, and particularly against the organisms most commonly found in device associated infections. Further work will be needed to investigate effectiveness of phage therapy on polymicrobial biofilms.

In conclusion, treatment of central venous catheter material with a bacteriophage antimicrobial-lock technique significantly reduced bacterial colonization and biofilm presence in an in-vitro model. Though evidence supporting the use of phages for the treatment of device-associated biofilms in humans is lacking, this and other recent studies involving the interaction of phage and biofilms have shown promise as an alternative therapy for the treatment of biofilm-associated infection and suggests that further investigation is warranted.

Material and Methods

Microorganisms and culture conditions

Staphylococcus aureus 46106, a methicillin-susceptible isolate from an abdominal wound that was negative for toxic shock syndrome toxin and Panton-Valentine leucocidin, obtained from the CDC Clinical and Environmental Laboratory Branch culture collection, was used for growing biofilms. Cultures were stored at −71 °C and subcultured on trypticase soy agar containing 5% sheep’s blood (blood agar) (BD Diagnostics) overnight and grown in Brain Heart Infusion Broth (BD Diagnostics) at 37 °C with shaking to obtain a cell suspension equivalent to a 0.5 MacFarland standard (10⁸ CFU/ml) on the day of use.

Phage strain selection

Staphylococcus aureus phage K and its host strain Staphylococcus aureus (ATCC 19685) were obtained from ATCC. Phage K was propagated using the soft agar overlay technique to titer levels of 10⁸ PFU.²⁷ Crude high titer phage broth cultures were prepared according to Adams²⁸ using Brain Heart Infusion Broth supplemented with 3 mM MgCl₂ and 4 mM CaCl₂ (added as MgCl₂ • 6 H₂O and CaCl₂ • 2 H₂O). Phage broth cultures were filter sterilized (0.2 µ) prior to use.

Experimental approach

Twenty 1 cm² silicone discs were arranged into separate 3 ml sterile wells filled with 2 mL of 10⁸ CFU/ml log phase culture of S. aureus 46106. The inoculum remained in place for 24 h. After 24 h the inoculum was carefully withdrawn from all wells by aspiration. The 20 discs were randomized to treatment or control arms. Following 24 h of bacterial inoculation, 10 control-arm discs were bathed in sterile phosphate buffered saline (PBS) and 10 experimental-arm discs which were bathed in 2 mL of a 10⁸ plaque forming units/ml (PFU/ml) solution of phage K, prepared as described above, for 24 h.

Silicone discs were removed for microbiological evaluation using aseptic technique, and processed to recover microorganisms using a modified previously published method.²⁰ Briefly the discs were rinsed gently in sterile PBS and placed into a tube containing 10 ml of PBS and subjected to three alternating 30 s cycles of water bath sonication (45 kHZ, Branson Water Bath Sonicator) and vortexing. The resulting biofilm suspension was diluted in Butterfield buffer, spread plated on Blood agar, incubated for 48 h at 37 °C and counted.

Statistical analysis

All statistical tests were performed using the Statistical Package for Social Sciences version 19 (SPSS). Mean plate counts (as log₁₀ CFU per cm²) for treated and untreated discs were compared using the Mann-Whitney U Test. A P value of < 0.05 was considered statistically significant.

Citation: Christensen D, Kankotia R, Falk I, Paxton B, Kim C, Lungren M. Bacteriophage K for reduction of Staphylococcus aureus biofilm on central venous catheter material. Bacteriophage 2013; 3:e26825; 10.4161/bact.26825

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.