Mechanically ventilated patients are at risk of contracting bacterial infections, most notably ventilator-associated pneumonia (VAP). In light of this, patients often receive parenteral antibiotics in response to early signs of inflammation or even as means of prevention for at-risk patients (1). At least for Staphylococcus aureus, a frequent contributor to infection in this setting (2,3), systemic antibiotic administration does not effectively reduce the prevalence of VAP, likely due to the limitations of anti-staphylococcal antibiotics, such as poor lung penetration and limited effectiveness against bacterial biofilms (1,4,5). Additionally, topical administration of antibiotics and disinfectants have produced mixed results and have been associated with the spread of antibiotic resistance (5). Clearly, antibiotic alternatives warrant investigation for the prevention of VAP due to S. aureus.
One strategy is the application of bacterial viruses known as phage therapy, which has been rediscovered for the treatment of many diverse, antibiotic-resistant infections (for a recent review, see reference ). In the context of VAP, intravenously administered phages have been shown to be as effective as standard of care antibiotics for the treatment of pneumonia due to methicillin-resistant S. aureus (MRSA) in an experimental model of VAP in rats (7). Phages have the added benefits of effectiveness against bacterial biofilms, and mechanisms of action that are distinct from typical antibiotics, which limits the threat of antibiotic resistance (8). Important questions relating to phage therapy remain, including when, how and how many bacteriophages should be delivered (8,9). In the current report, we have assessed the utility of nebulized bacteriophages (“aerophages”) for prophylaxis of VAP due to MRSA in an experimental rat model.
MATERIALS AND METHODS
All animal experiments were approved by the Cantonal Committee on Animal Experiments of the State of Bern, Switzerland (approval BE 53/19). We first addressed the bioavailability of aerophages over a 24-hour period in uninfected Wistar rats (male, 300–400 g). Twelve animals were administered 3 mL of a multiphage cocktail (consisting of phages K, 3A, 2002 and 2003 at final concentration of 1 × 1010 plaque forming units [PFUs] per mL) using a modified vibrating mesh aerosol drug delivery system (Aerogen Solo, Aerogen, Ireland). Animals anesthetized with sevoflurane (Fi 1–3%) were connected to the nebulizer via a facemask and aerophages were delivered inside an enclosed induction chamber. Following aerophage application, animals were returned to their cages. Animals were euthanized at 0, 2, 6, and 24 hours (n = 3 per time point) at which point the lungs were homogenized and phages were quantified in double-layer agar plates as described previously (7). To determine if increasing the number of aerophages would improve experimental outcomes, the phage cocktail was concentrated via isopycnic ultracentrifugation through a caesium chloride (CsCl2) gradient, the residual CsCl2 was removed via filtration (Amicon Ultra Centrifugal Filters; Merck, Darmstadt, Germany), and the phages were resuspended in 0.9% sodium chloride to a final concentration of 1 × 1011 PFU/mL. To address bioavailability, animals (n = 4) were administered 3 mL of the 1 × 1011 PFU/ml cocktail, euthanized, and phages in the lungs were quantified as described above.
We next employed our established model of severe MRSA VAP essentially as described elsewhere (7). Briefly, animals (n = 10 per treatment group) received a single dose of either 3 × 1010 PFU or 3 × 1011 PFU of aerophages as described above and were returned to their cages. After 2 hours, animals were ventilated for 4 hours on ventilators for small animals (VentElite; Harvard Apparatus [Holliston, MA]; 10 mL/kg tidal volume, 5 cm H2O of positive end-expiratory pressure, 50 breaths/min with Fio2 0.35). Anesthesia was maintained with inhaled sevoflurane (Fi 3–4%) with additional subcutaneous fentanyl (20 µg/kg) delivered every 30–60 minutes. A total of 6 hours after aerophage prophylaxis, animals were inoculated with ~8 × 109 colony forming units of an alpha-toxin producing clinical MRSA strain (AW7) via the endotracheal tube and were then extubated. Animal welfare was determined at least three times per day over the course of 96 hours, and the severity of illness was scored using an animal welfare scoring system (Supplemental Table 1, Supplemental Digital Content 1, http://links.lww.com/CCM/F449). Animals were euthanized with pentobarbital (150 mg/kg) if they scored greater than “1,” or if their weight decreased by more than 20% (classed as “non-survivors”) (7). Remaining animals were euthanized at 96 hours and classed as “survivors.” Quantification of MRSA burdens in the lungs and spleen was performed following tissue homogenization as described previously (7). Histopathological analyses were performed as described previously (7), and a histopathological score was calculated for lung tissues according to parameters detailed in Supplemental Table 2 (Supplemental Digital Content 1, http://links.lww.com/CCM/F449). Infected and untreated animals (n = 7) were included as controls to validate the lethality of the model.
A single application of 3 × 1010 PFU of aerophages to the airways of uninfected rats resulted in moderate phage loads in the lungs immediately after administration (1 × 106 PFU/g; Fig. 1A), and a phage lung burden was still apparent at 24 hours (4 × 104 PFU/g; Fig. 1B). Application of a higher dose (3 × 1011 PFU) to uninfected animals resulted in an average phage load for the lungs of 6 × 106 PFU/g soon after application (Fig. 1A).
In the context of experimental VAP due to MRSA, all control animals showed severe symptoms and succumbed to infection within 48 hours (Fig. 1C). A single application of 3 × 1010 PFU of aerophages prior to ventilation and infection prevented mortality in 60% of animals (Fig. 1C; p < 0.01 compared with untreated controls; log-rank test). Administration of 3 × 1011 PFU prior to ventilation rescued 70% of animals, which was higher than for untreated animals (p < 0.01; log-rank test), but survival was not statistically different compared with those that received the lower dose of phages (p = 0.66).
Mortality in experimental models of VAP due to MRSA correlates with high bacterial loads in the lungs (7). In support of this, surviving animals treated with prophylactic aerophages had ~500 times less MRSA in the lungs compared with either untreated controls, or treated animals that succumbed to infection (Fig. 2A; p < 0.01 using one-way analysis of variance [ANOVA]; and p < 0.01 for each comparison using Tukey multiple comparisons test). When lung bacterial loads were compared between control animals and each phage treatment group, no significant differences were determined, likely owing to the high MRSA loads for the nonsurviving animals present in each group (Fig. 2B; p = 0.17; one way ANOVA). There was a positive correlation between bacterial burden in the lungs and histopathological scoring of the lung tissue for animals that received phage prophylaxis (Fig. 2C; r = 0.80; p < 0.0001; Pearson two-tailed correlation test). MRSA loads for the spleen, which we use as a marker for the systemic spread of infection, were not different between surviving and nonsurviving animals (Fig. 2D; p = 0.24; one-way ANOVA) or between treatment groups (Fig. 2E; p = 0.88; one-way ANOVA). Phages were recovered from the lungs of most of the phage treated animals after euthanasia (17/20; Fig. 2F), and were infrequently detected in the spleen (1/20 animals) suggesting aerophages remain localized in the lung tissue and do not readily spread to other organs.
DISCUSSION AND CONCLUSIONS
A single, prophylactic application of a nebulized phage cocktail prior to ventilation prevented lethal infection for the majority of animals in an experimental model of VAP due to MRSA (Fig. 1). Survival was associated with substantial decreases in bacterial burden in the lungs and reduced lung tissue damage (Fig. 2). The results of the current report provide an important proof-of-concept for phage prophylaxis in the context of VAP. Although we observed promising results for laboratory animals in a controlled study, the distribution of phages and effectiveness of prophylaxis in severely damaged and heterogeneously ventilated lungs of human patients remains to be determined.
Phage prophylaxis joins a small but expanding list of appealing antibiotic alternatives that may be effective for VAP prevention. Generally, probiotics have shown promise in mechanically ventilated patients, whereby inoculation with Lactobacillus proved to be effective at reducing the prevalence of VAP in a placebo-controlled trial, irrespective of the causative organism (10). When focusing on MRSA, monoclonal antibodies that can neutralize S. aureus toxins have shown promise for the prevention of pneumonia in animal models (11), they are safe and well-tolerated in healthy human adults (12), and are being investigated in an interventional clinical trial for the prevention of pneumonia in high-risk patients (NCT02296320).
Our current findings pave the way for future phase I/II trials that will address safety, dosing and tolerability of phages in patients at risk of MRSA VAP including those colonized with S. aureus, and expected to require prolonged ventilation (i.e., longer than 48 hr).
We acknowledge Sandra Nansoz and Robert Lukesch for outstanding technical assistance, and Sara Soto from the Institute of Animal Pathology, Vetsuisse Faculty, University of Bern, Switzerland for performing histopathological analyses.
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