Development and Use of Personalized Bacteriophage-Based Therapeutic Cocktails To Treat a Patient with a Disseminated Resistant Acinetobacter baumannii Infection

Abstract

Widespread antibiotic use in clinical medicine and the livestock industry has contributed to the global spread of multidrug-resistant (MDR) bacterial pathogens, including Acinetobacter baumannii We report on a method used to produce a personalized bacteriophage-based therapeutic treatment for a 68-year-old diabetic patient with necrotizing pancreatitis complicated by an MDR A. baumannii infection. Despite multiple antibiotic courses and efforts at percutaneous drainage of a pancreatic pseudocyst, the patient deteriorated over a 4-month period. In the absence of effective antibiotics, two laboratories identified nine different bacteriophages with lytic activity for an A. baumannii isolate from the patient. Administration of these bacteriophages intravenously and percutaneously into the abscess cavities was associated with reversal of the patient's downward clinical trajectory, clearance of the A. baumannii infection, and a return to health. The outcome of this case suggests that the methods described here for the production of bacteriophage therapeutics could be applied to similar cases and that more concerted efforts to investigate the use of therapeutic bacteriophages for MDR bacterial infections are warranted.

Keywords: Acinetobacter; bacteriophage therapy; multidrug resistance.

FIG 1

Clinical course before and during the initial phase of bacteriophage therapy. Positive blood cultures are depicted above the graphic data, whereas antibiotic and phage administration are indicated below.

FIG 2

Screening of A. baumannii phage library against TP1 clinical isolate. A subset of 98 phages from the Navy phage library were individually tested against the A. baumannii TP1 strain using the OmniLog system (Biolog, Hayward, CA) as described in references and . Briefly, growth of bacteria or lack of growth due to lysis from phage infection was monitored every 15 min via a redox chemical reaction employing cellular respiration as a universal reporter, where cellular respiration from growth reduces a tetrazolium-based dye and produces a color change. If the growth is weakly positive or is negative, then respiration is slow or absent and so little to no color change is observed. The results of this assay after 20 h are summarized here, where the color gradient indicates the duration of bacterial growth inhibition.

FIG 3

Activity of bacteriophage cocktails. Activity of cocktails ΦPC and ΦIV against serial isolates of A. baumannii isolated from intra-abdominal drains before bacteriophage therapy (strain TP1) (a and e), 4 days after initiation of antibiotic therapy (strain TP2) (b and f), and 8 days after initiation of bacteriophage therapy (strain TP3) (c and g). Panel d demonstrates the derivation of a second-generation bacteriophage cocktail directed at the TP3 A. baumannii strain, a mixture of AB-Navy71 and AbTP3Φ1. Panel h demonstrates the additive activity of the ΦIV bacteriophage cocktail (10⁷ PFU) and a sublethal concentration of minocycline (0.25 μg/ml) against A. baumannii strain TP3. The 50% inhibitory concentrations of minocycline for A. baumannii strains TP1, TP2, and TP3 were 1, 2, and 4 μg/ml, respectively.

FIG 4

Bacteriophage titer from plasma samples during bacteriophage therapy. Plasma samples collected 5 min prior to and following administration of 5 × 10⁹ PFU of bacteriophage via intravenous injection indicated that bacteriophage titers in systemic circulation increase rapidly from 0 PFU/ml to 1.8 × 10⁴ PFU/ml. The bacteriophage titer dropped to 4.4 × 10³ PFU/ml by 30 min, 3.3 × 10² PFU/ml by 60 min, and 20 PFU/ml by 120 min postinjection. Plasma samples collected 6 h following the initial injection contained no detectable bacteriophage titer (limit of bacteriophage detection, 20 PFU/ml).

FIG 5

Transmission electron micrographs of A. baumannii-specific bacteriophages. Electron micrographs of the ΦIV cocktail phages showed large prolate myobacteriophage morphology (a to d) and short small podophage morphology (e). Bacteriophage AbTP3Φ1, isolated against strain TP3, is a small podophage. (a) AB-Navy1; (b) AB-Navy4; (c) AB-Navy71; (d)AB-Navy97; (e) AbTP3Φ1.

FIG 6

Investigation into morphological characteristics of A. baumannii isolates. The apparent morphological differences of the three A. baumannii isolates were examined by Raman spectroscopy using a PhAT Probe 830-nm system (a) and by capsule staining with crystal violet (b to d). Capsule staining of isolates TP1 (b), TP2 (c), and TP3 (d) is shown.