Personalized aerosolised bacteriophage treatment of a chronic lung infection due to multidrug-resistant Pseudomonas aeruginosa

Abstract

Bacteriophage therapy has been suggested as an alternative or complementary strategy for the treatment of multidrug resistant (MDR) bacterial infections. Here, we report the favourable clinical evolution of a 41-year-old male patient with a Kartagener syndrome complicated by a life-threatening chronic MDR Pseudomonas aeruginosa infection, who is treated successfully with iterative aerosolized phage treatments specifically directed against the patient's isolate. We follow the longitudinal evolution of both phage and bacterial loads during and after phage administration in respiratory samples. Phage titres in consecutive sputum samples indicate in patient phage replication. Phenotypic analysis and whole genome sequencing of sequential bacterial isolates reveals a clonal, but phenotypically diverse population of hypermutator strains. The MDR phenotype in the collected isolates is multifactorial and mainly due to spontaneous chromosomal mutations. All isolates recovered after phage treatment remain phage susceptible. These results demonstrate that clinically significant improvement is achievable by personalised phage therapy even in the absence of complete eradication of P. aeruginosa lung colonization.

Fig. 1. Patient treatment, follow-up and sample collection.

a Antibiotic treatments are indicated by green horizontal lines and phage therapies by blue vertical lines. P. aeruginosa isolates selected for genotypic and phenotypic analysis are shown above the black-dotted vertical lines. Numbering of isolates and events is in reference to the day of the first phage treatment onset (D0). Episodes of exacerbations are indicated by red dots. Hospital and rehabilitation stays are indicated on the horizontal bar. b Computed tomography (CT) lung scans were performed at the days indicated, with respect to phage treatment onset (D0).

Fig. 2. Bacterial and phage load in sputum samples during and between phage courses.

Samples are numbered with respect to the first phage administration (D0). The first sputum sample was taken 15 min before the first phage dose, while all other samples were obtained 7–8 h after phage administration. a Sputum samples were analyzed for the presence of P. aeruginosa, A. xylosoxidans as well as phage DNA by qPCR on total genomic DNA. b In parallel, combined viable counts of P. aeruginosa and A. xylosoxidans (CFU) were determined on cetrimide agar plates in triplicate, which do not allow distinguishing between these bacterial species. Infective phage particles (PFU) were counted on phage indicator plates in triplicate. LoQ limit of quantification, LoD limit of detection. Phage treatment courses are indicated by gray-shaded rectangles.

Fig. 3. Susceptibility of P. aeruginosa isolates to phage vFB297 and beta-lactam antibiotics.

Transmission electron microscopy images of (a) Pakpunavirus vFB297 (top panel) and susceptible D0.4 bacterial cell lysed by vFB297 phage particles (bottom panel). b, c Susceptibility of patient isolates to phage vFB297 was determined by plaque assays (for full dataset, see Supplementary Fig. S2). S susceptible (plaques visible in phage dilutions), I intermediate (plaques visible only in undiluted phage), R resistant (no plaques visible). d–f Susceptibilities to beta-lactam antibiotics of P. aeruginosa isolates from consecutive sputum samples. MIC determinations were performed on three occasions, yielding similar results. MIC breakpoints (EUCAST 2019) for beta-lactams are shown by dashed lines. Each symbol represents a single P. aeruginosa isolate. The seven isolates selected for WGS are represented by closed symbols. Phage treatment courses are indicated by gray-shaded rectangles.

Fig. 4. Phylogeny of patient isolates.

All isolates sequenced belong to the same DLST-type (15-29). Isolates D0.1, D6.1, and D467 (shown in bold) were isolated during the first and second phage treatment, respectively. Phylogeny results are based on SNP analysis using vcf2phylip v2.6 and FigTree v1.4.4. Numbers in parentheses indicate the number of additional cumulative SNPs compared to the D-100 isolate as determined by WGS (Supplementary Dataset 1). Bootstrap values are shown at the branching points.

Fig. 5. Cytotoxicity and virulence of P. aeruginosa isolates.

a Calu-3 cells were infected with PAO1 or patient isolates and trans-epithelial resistance was determined at 24 h p.i. and 72 h p.i. Values represent mean and standard error of the mean (SEM) of three independently performed experiments in technical duplicates. b PAO1 and patient isolates were tested for their virulence in a Galleria mellonella infection model. The Kaplan–Meier plots represent the survival of 20–30 larvae that were used for each strain. Larvae survival was followed up to 72 h. PAO1 was the virulent reference strain (>90% killing at 72 h p.i.). The significance of differences of TER (a) or larvae survival (b) were assessed using one-way ANOVA with Dunnett multi-comparison test (a) or log-rank test (b), by comparison to the positive control strain PAO1.