Treatment protocol

This cohort of eight female and one male pwCF, median age 32 years (range 22–46 years), were the first nine patients treated with compassionate phage therapy because of MDR (seven patients) or PDR (two patients) PsA that did not respond to standard cystic fibrosis (CF) therapies (Table 1). All patients had a clinical course complicated by frequent pulmonary exacerbations despite oral, inhaled and/or intravenous (IV) antibiotics without evidence of clinical benefit. When phage therapy was initiated, all patients were concurrently on IV antibiotics (n = 6) or recently completed antibiotics (n = 3; Table 2). Some patients had additional sputum pathogens (for example, Staphylococcus aureus, Achromobacter spp. and nontuberculous mycobacteria; Table 1). At the time of phage therapy, elexacaftor–tezacaftor–ivacaftor (Trikafta or Kaftrio) was not available. Two individuals were taking tezacaftor–ivacaftor (Symdeko or Symkevi; Table 1). Individuals were selected for treatment owing to a combination of the following: lack of clinical response to antibiotics, persistent symptoms and/or clinical decline despite comprehensive CF care, severity of pulmonary exacerbations, and no additional approved therapeutics available.

Phage therapy was personalized on the basis of susceptibility of the PsA sputum isolates to phages in the phage library (Table 2) at Yale’s Center for Phage Biology and Therapy, which provided phages for all participants. After suitable phage(s) were identified, a treatment protocol was reviewed with each participant’s CF physician. This protocol, phage manufacturing and the informed consent form were reviewed and approved via US Food and Drug Administration (FDA) single patient investigational new drug (SPIND) and local institutional review boards. Each participants provided written informed consent.

Inhaled phage therapy was given twice daily for inpatients (n = 4) or daily for outpatients (n = 5) for 7–10 days. Inpatients were provided jet nebulizers; outpatients used their own jet nebulizers (Table 2). Phages were delivered as mixtures (cocktails) of two or three phages (n = 6) or single-phage therapy (n = 3; Table 2), administered at a total dose of 1 × 10¹⁰ plaque-forming units (PFU). Phage nebulization was well tolerated without any adverse events. For the five outpatients, four reported subjective fevers and three reported fatigue on days 2 and 3 of phage therapy, without the need for additional treatment(s). The four inpatients did not have documented fever or report symptoms after phage therapy.

Inhaled phage therapy decreases sputum PsA

To test the hypothesis that phage therapy would reduce sputum PsA in each participant, spontaneously expectorated sputum collected before and after therapy was processed for PsA colony-forming unit (CFU) quantification. Sputum PsA CFU before therapy was greater than after therapy for all participants (Fig. 1a and Table 2). Across all participants, sputum PsA CFUs decreased after phage therapy from a median (first quartile, third quartile) 2.6 × 10⁸ (5.0 × 10⁷, 5.7 × 10⁸) or mean 3.0 × 10⁸ (±1.0 × 10⁸ s.e.m.) CFU ml⁻¹ before therapy, to median 2.6 × 10⁴ (1.2 × 10⁴, 3.1 × 10⁶) or mean 7.7 × 10⁶ (±6.9 × 10⁶ s.e.m.) CFU ml⁻¹ after completion of therapy (5–18 days (median 7, mean 8.4)), which is a 10⁴ median or 10² mean difference, respectively (P = 0.006, two-way analysis of variance (ANOVA) with Dunnett’s multiple-comparisons test). A later time point (15–42 days (median 20, mean 22.7)) after completion of phage therapy found decreased PsA CFU median 7.8 × 10⁵ (2.0 × 10⁴, 2.7 × 10⁷) or mean 1.1 × 10⁷ (±7.5 × 10⁶ s.e.m.) CFU ml⁻¹, which is a 10³ median, or 10¹ mean difference, respectively (P = 0.0112, two-way ANOVA with Dunnett’s multiple-comparisons test). Thus, sputum PsA decreased after phage therapy in each patient and across the cohort, regardless of individualized differences in the phage treatment strategies (Fig. 1a and Table 2).

a, Sputum analysis was performed before and after phage therapy. PsA CFU ml⁻¹ from each patient’s sputum (n = 9) were counted in replicates of three and averaged. CFU ml⁻¹ was measured before therapy (Pre) and at two time points after therapy (Post (14 days), 5–18 days (average 8.4, median 7), and Post (30 days), 15–42 days (average 22.7, median 20)); **P = 0.006 and *P = 0.0112; two-way ANOVA with Dunnett’s multiple-comparisons test. Two participants did not provide sputum samples after Post (14 days). b, Spirometry was performed before and after (days 21–35) phage therapy and reported as ppFEV1% best single measurement from at least three tests per the American Thoracic Society standards of acceptability and repeatability; **P = 0.004; Wilcoxon signed-rank two-tailed t-test. PT, patient.

Inhaled phage therapy improves lung function

In CF the most salient outcome is lung function (also known as spirometry), and therapies that decrease PsA may be associated with this clinical benefit¹¹. Therefore, we compared lung function (percent predicted forced expiratory volume in 1 s (ppFEV₁)) before versus after phage therapy (days 21–35). Results for each participant showed an increase in ppFEV₁ after versus before therapy (Table 2 and Fig. 1b), and an analysis across the entire cohort showed an overall improvement in ppFEV₁ from a median (first quartile, third quartile) 36 (23, 49), or mean 37 ± 5.5 s.e.m., before therapy to a median 42 (27, 67), or mean 45 ± 6.9 s.e.m., after therapy, which is median or mean ppFEV1 difference of 6 and 8, respectively (P = 0.004, Wilcoxon signed-rank two-tailed t-test). Analysis of participants with pre-therapy ppFEV₁ >30 showed increased ppFEV₁ after phage therapy (mean difference of 11 ± 3.2 s.e.m. (P = 0.06) Wilcoxon signed-rank two-tailed t-test), while participants with pre-therapy ppFEV₁ <30 improved ppFEV₁ to a lesser extent (mean difference 3.5 ± 0.96 s.e.m. (P = 0.1) Wilcoxon signed-rank two-tailed t-test). Therefore, phage therapy was associated with improved lung function, regardless of individualized differences in phage treatment strategies (Table 2 and Fig. 1b).

Phage therapy reduces PsA virulence

In addition to bacterial killing (Fig. 1a), we hypothesized that our personalized strategy to use specific phages to target AMR or virulence factors should select for surviving bacterial mutants with evolved phage resistance that results in decreased virulence. First, PsA resistance to phage therapy was investigated by studying purified PsA clones from participant sputum, and each isolate was tested for susceptibility to phage(s) used in inhaled therapy (Table 2). For each participant, results showed that post-therapy sputum PsA contained one or more bacterial clones with resistance to phage(s) used in therapy (Table 2), which showed that inhaled phage therapy exerted selection for PsA to evolve resistance to phage(s) used in treatment.

Next, we examined whether evolved phage resistance after therapy coincided with predicted trade-offs with antibiotic resistance or virulence. Phage OMKO1 selects for evolved phage resistance that coincides with increased sensitivity to various antibiotics^9,10, which may be explained by this phage binding to Mex efflux pumps^9,10. In the two patients who received OMKO1 (Table 2), post-therapy sputum isolates evaluated in clinical laboratories for antibiotic sensitivity showed PsA with increased susceptibility to imipenem (patient 1; Fig. 2a) and piperacillin–tazobactam, ceftazidime, amikacin, gentamicin and tobramycin (patient 3; Fig. 2b). Additional pre- and post-therapy PsA isolates were examined in our research laboratory for changes in susceptibility (minimum inhibitory concentration; MIC) to antibiotics. For patients who did not receive OMKO1, fewer PsA isolates showed changes to antibiotic susceptibility (Extended Data Fig. 1b,d–i). These results suggest that the predicted resensitivity to antibiotics occurred in the two patients who received OMKO1 (Fig. 2a,b). As expected, patients who did not receive OMKO1 did not show antibiotic resensitization in most isolates (Extended Data Fig. 1).

a,b, Antibiotic susceptibility results from clinical microbiology laboratory testing for sputum isolates taken before and after therapy from patient 1 (a) and patient 3 (b) are shown (R, resistant; S, susceptible; I, intermediate; X, not reported). c, Production of pyocyanin (µg ml⁻¹) was measured from PsA isolates before therapy (N = 6) and after (N = 10) therapy from patients who received TIVP-H6 phage therapy (**P = 0.0047; Mann–Whitney test). d, Attachment of PsA to CF airway epithelial cells before (N = 6) and after (N = 6) therapy in duplicate from patient 2 (***P = 0.0005; Mann–Whitney test). e, LPS (ng CFU⁻¹) quantification from PsA sputum isolates taken before therapy (N = 14) and after therapy (N = 25) from patients who received phage therapy with LPS-5 (P = 0.6963; Mann–Whitney test). f, Secreted elastase activity (U ml⁻¹) from PsA sputum isolates taken before therapy (N = 8) and after therapy (N = 15) from patients who received phage therapy with LPS-5 (**P = 0.002; Mann–Whitney test). Data presented as mean values ± s.d.

The function of PsA TIVP includes twitching motility, secretion of pyocyanin (a virulence factor that causes cellular inflammation and oxidative stress¹²) and bacterial surface attachment¹³. Phage TIVP-H6 binds to PsA TIVP, and we investigated the hypothesis that TIVP-H6 phage therapy selects against TIVP function. Pyocyanin was measured from PsA isolates before and after therapy. Results showed that pre-therapy isolates from patients 1, 3, 4, 5, 6, 8 and 9 showed minimal to no pyocyanin production, while isolates from patients 2 and 7 secreted pyocyanin at detectable levels before therapy (Fig. 2c and Extended Data Fig. 2a–i). However, only isolates from patient 7 showed a statistically significant reduction of pyocyanin production (Extended Data Fig. 2b,g). Finally, the potential for phage TIVP-H6 to select for reduced bacterial adherence was studied in vitro using CF airway epithelial (CFBE41o-) cells grown at an air–liquid interface (ALI) using isolates from patient 2 (the only patient to receive single-phage TIVP-H6 therapy). Compared with pre-therapy PsA, post-therapy isolates showed significantly decreased adherence to CF airway epithelium (Fig. 2d). Together these results show that TIVP-H6 phage therapy selects for a trade-off in post-therapy PsA isolates for reduced virulence via a reduction in pyocyanin (Fig. 2c) and/or decreased TIVP-mediated adherence to CF airway epithelium (Fig. 2d).

PsA LPS contributes to virulence by reducing antibiotic permeability, contributing to biofilm production and activating human immunity¹⁴. As phage LPS-5 binds to PsA LPS, LPS per cell (Fig. 2e and Extended Data Fig. 3) and elastase production (Fig. 2f and Extended Data Fig. 4) from pre- and post-therapy PsA isolates were measured. Across all eight patients who received phage LPS-5, there was no statistically significant change in LPS content before versus after therapy (Fig. 2e). A similar analysis for elastase activity showed a significant decrease in elastase activity across all patients who received LPS-5 and produced elastase before therapy (Fig. 2f); patients 1 and 6 (Extended Data Fig. 4a,f) had decreased elastase activity after phage therapy. While evidence for phage LPS-5-selected trade-offs was mixed, these results show that LPS-5 phage therapy selects for a trade-off in post-therapy PsA isolates for reduced elastase production (Fig. 2f).

Phage therapy does not change bacterial species diversity

Because antibiotics have secondary effects on nontarget bacteria, a similar possibility can be examined for administered phage(s). Specifically, reducing PsA in the lung may open niche(s) for other species, which were previously occupied by PsA. This is particularly important in pwCF where several pathogens can contribute to lung disease^15,16. Thus, potential off-target effects were investigated in longitudinal sputum samples obtained before and after therapy. First, results from clinical laboratory sputum cultures showed no change in CF pathogens before or after therapy. Second, metagenomic analysis of longitudinal changes in alpha-diversity (species richness)¹⁷ present in pre- and post-therapy sputum from patients 2–9 (Extended Data Fig. 5a) showed no significant change(s) in: (1) Chao1 diversity index (Extended Data Fig. 5b), which is a richness metric that especially considers potentially missing (‘rare’) species¹⁸; (2) Shannon diversity index (Extended Data Fig. 5c), which accounts for the abundance and evenness of species across time¹⁹; and (3) Simpson diversity index (Extended Data Fig. 5d), which accounts for the presence of specific dominant species, aside from overall abundance²⁰. Thus, these results showed that phage therapy did not significantly alter the composition of bacterial communities in these patients.

Pre- and post-therapy PsA strains are closely related

While the above results show that phage therapy selects for phage resistance commensurate with trade-offs in antibiotic resistance or bacterial virulence at a phenotypic level, we hypothesized that we could detect similar signal(s) at the genotypic level. Whole-genome sequencing and variant analysis of the pre- and post-therapy isolates showed that post-therapy isolates had numerous mutations not found in pre-therapy isolates (Fig. 3 and Extended Data Fig. 6). For example, patient 1 post-therapy isolate showed nonsynonymous mutations in the sequences encoding MexB (R586S), a component of a complex involved in efflux of several antibiotics; MigA (P169T), an enzyme involved in LPS biosynthesis; and multiple proteins involved in TIVP assembly, one of which (PilE P81R) was shared by all post-therapy isolates examined (Fig. 3a). Patient 4 had nonsynonymous mutations in two LPS biosynthesis genes, wzm and PA5455 (Fig. 3B) in four out of five post-therapy isolates, which might account for the lower LPS content observed in these isolates (Extended Data Fig. 3). Several mutations involved in TIVP biosynthesis were observed in all post-therapy isolates from patient 7, which correlate with the significantly lower pyocyanin measured from these isolates (Extended Data Fig. 2), although only one isolate showed a mutation in a LPS biosynthesis gene (Fig. 3c). Many mutations in isolates from these patients, as well as the other six patients (Extended Data Fig. 6), were found in genes related to the synthesis, modification or regulation of the receptors used by the phage(s) these patients received. However, nonsynonymous mutations were also found across isolate genomes at loci that do not obviously correspond with the phage treatments, which suggests that the PsA populations from which these isolates were drawn are genetically diverse and under in vivo selection pressures other than phage(s). A larger isolate set and more tailored laboratory experiments are needed to directly associate the mutations highlighted above with the measured phenotypes of interest.

a–c, Circular plots showing the distribution of variants in the post-treatment PsA isolate genomes of patient 1 (a), patient 4 (b) and patient 7 (c). Concentric circles represent single isolate genomes. Gray lines represent nonconservative variants that appear in coding sequences in one or more post-treatment isolates but are absent from all pre-treatment isolates. Orange lines represent nonconservative variants (nonsynonymous polymorphisms and frameshifts) coincident with genes expected to be under selection by the phage used in treatment. Labeled, colored arrows indicate the positions and functional categories of these genes. Vertical black lines represent the genome start in the PAO1 reference against which variants were called.